Acyl piperidine inhibitors of soluble epoxide hydrolase

ABSTRACT

Inhibitors of the soluble epoxide hydrolase (sEH) are provided that incorporate multiple pharmacophores and are useful in the treatment of diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/575,588 filed Feb. 12, 2013, now issued as U.S. Pat. No.9,296,693; which is a 35 USC §371 National Stage application ofInternational Application No. PCT/US2011/022901 filed Jan. 28, 2011, nowexpired; which claims the benefit under 35 USC §119(e) to U.S.Application Ser. No. 61/299,495 filed Jan. 29, 2010, now expired. Thedisclosure of each of the prior applications is considered part of andis incorporated by reference in the disclosure of this application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos. R01ES002710, P42 ES004699 and R01 ES013933 awarded by the NationalInstitute of Environmental Health Sciences. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Epoxide hydrolases (EHs, EC 3.3.2.3) catalyze the hydrolysis of epoxidesor arene oxides to their corresponding diols by the addition of water(see, Oesch, F., et al., Xenobiotica 1973, 3, 305-340). Some EHs play animportant role in the metabolism of a variety of compounds includinghormones, chemotherapeutic drugs, carcinogens, environmental pollutants,mycotoxins, and other harmful foreign compounds.

There are two well-studied EHs, microsomal epoxide hydrolase (mEH) andsoluble epoxide hydrolase (sEH). These enzymes are very distantlyrelated, have different subcellular localization, and have different butpartially overlapping substrate selectivities. The soluble andmicrosomal EH forms are known to complement each other in degrading someplant natural products (see, Hammock, B. D., et al., COMPREHENSIVETOXICOLOGY. Oxford: Pergamon Press 1977, 283-305 and Fretland, A. J., etal., Chem. Biol. Intereract 2000, 729, 41-59).

The major role of the sEH is in the metabolism of lipid epoxidesincluding the metabolism of arachidonic acid (see, Zeldin, D. C., etal., J. Biol. Chem. 1993, 268, 6402-6407), linoleic acid (see,Moghaddam, M. F., et al., Nat. Med. 1997, 3, 562-567) acid, some ofwhich are endogenous chemical mediators (see, Carroll, M. A., et al.,Thorax 2000, 55, SI 3-16). Epoxides of arachidonic acid(epoxyeicosatrienoic acids or EETs) and other lipid epoxides and diolsare known effectors of blood pressure (see, Capdevila, J. H., et al., J.Lipid. Res. 2000, 41, 163-181), and modulators of vascular permeability(see, Oltman, C. L., et al., Circ. Res. 1998, 83, 932-939). Thevasodilatory properties of EETs are associated with an increasedopen-state probability of calcium-activated potassium channels leadingto hyperpolarization of the vascular smooth muscle (see Fisslthaler, B.,et al., Nature 1999, 401, 493-497). Hydrolysis of the arachidonateepoxides by sEH diminishes this activity (see, Capdevila, J. H., et al.,J. Lipid. Res. 2000, 41, 163-181). sEH hydrolysis of EETs also regulatestheir incorporation into coronary endothelial phospholipids, suggestinga regulation of endothelial function by sEH (see, Weintraub, N. L., etal., Am. J. Physiol. 1992, 277, H2098-2108). It has recently been shownthat treatment of spontaneous hypertensive rats (SHRs) with selectivesEH inhibitors significantly reduces their blood pressure (see, Yu, Z.,et al., Circ. Res. 2000, 87, 992-998). In addition, it was claimed thatmale knockout sEH mice have significantly lower blood pressure thanwild-type mice (see Sinai, C. J., et al., J. Biol. Chem. 2000, 275,40504-405010), however subsequent studies demonstrated with backbreeding into C57b mice that 20-HETE levels increased compensating forthe increase in plasma EETs (see, Luria, A. et al., J. Biol. Chem. 2007,282:2891-2898.

The EETs have also demonstrated anti-inflammatory properties inendothelial cells (see, Node, K., et al., Science 1999, 285, 1276-1279and Campbell, W. B., Trends Pharmacol. Sci. 2000, 21, 125-127). Incontrast, diols derived from epoxy-linoleate (leukotoxin) perturbmembrane permeability and calcium homeostasis (see, Moghaddam, M. F., etal., Nat. Med. 1997, 3, 562-567), which results in inflammation that ismodulated by nitric oxide synthase and endothelin-1 (see, Ishizaki, T.,et al., Am. J. Physiol. 1995, 269, L65-70 and Ishizaki, T., et al., J.Appl. Physiol. 1995, 79, 1106-1611). Micromolar concentrations ofleukotoxin reported in association with inflammation and hypoxia (see,Dudda, A., et al., Chem. Phys. Lipids 1996, 82, 39-51), depressmitochondrial respiration in vitro (see, Sakai, T., et al., Am. J.Physiol. 1995, 269, L326-331), and cause mammalian cardiopulmonarytoxicity in vivo (see, Ishizaki, T., et al., Am. J. Physiol. 1995, 269,L65-70; Fukushima, A., et al., Cardiovasc. Res. 1988, 22, 213-218; andIshizaki, T., et al., Am. J. Physiol. 1995, 268, L123-128). Leukotoxintoxicity presents symptoms suggestive of multiple organ failure andacute respiratory distress syndrome (ARDS) (see, Ozawa, T. et al., Am.Rev. Respir. Dis. 1988, 137, 535-540). In both cellular and organismalmodels, leukotoxin-mediated toxicity is dependent upon epoxidehydrolysis (see, Moghaddam, M. F., et al., Nat. Med. 1997, 3, 562-567;Morisseau, C., et al., Proc. Natl. Acad. Sci. USA 1999, 96, 8849-8854;and Zheng, J., et al., Am. J. Respir. Cell Mol. Biol. 2001, 25,434-438), suggesting a role for sEH in the regulation of inflammationand vascular permeability. The bioactivity of these epoxy-fatty acidssuggests that inhibition of vicinal-dihydroxy-lipid biosynthesis mayhave therapeutic value, making sEH a promising pharmacological target.

Recently, 1,3-disubstituted ureas, carbamates, and amides have beenreported as new potent and stable inhibitors of sEH. See, U.S. Pat. No.6,150,415. Compounds 192 and 686 are representative structures for thistype of inhibitors (FIG. 1, therein). These compounds are competitivetight-binding inhibitors with nanomolar K_(I) values that interactstoichiometrically with purified recombinant sEH (see, Morisseau, C., etal., Proc. Natl. Acad. Sci. USA 1999, 96, 8849-8854). Based on the X-raycrystal structure, the urea inhibitors were shown to establish hydrogenbonds and to form salt bridges between the urea function of theinhibitor and residues of the sEH active site, mimicking featuresencountered in the reaction coordinate of epoxide ring opening by thisenzyme (see, Argiriadi, M. A., et al., Proc. Natl. Acad. Sci. USA 1999,96, 10637-10642 and Argiriadi, M. A., et al., J. Biol. Chem. 2000, 275,15265-15270). These inhibitors efficiently reduced epoxide hydrolysis inseveral in vitro and in vivo models (see, Yu, Z., et al., Circ. Res.2000, 87, 992-998; Morisseau, C., et al., Proc. Natl. Acad. Sci. USA1999, 96, 8849-8854; and Newman, J. W., et al., Environ. HealthPerspect. 2001, 109, 61-66). Despite the high activity associated withthese inhibitors, there exists a need for compounds possessing similaror increased activities, preferably with improved solubility and/orpharmacokinetic properties to facilitate formulation and delivery.

The present invention provides such compounds along with methods fortheir use and compositions that contain them.

SUMMARY OF THE INVENTION

In one aspect, the present invention provide a compound of formula II:

wherein each R¹ of formula II is independently H, halogen, C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, —O-aryl, heterocycloalkylhaving 5-6 ring members and at least 1 N heteroatom and 1 O heteroatomas ring members, —OH, —NO₂ or —C(O)OR³, wherein at least 1 R¹ is otherthan H. Alternatively, two R¹ groups on adjacent carbons are joined toform a 5-6 membered heterocycloalkyl ring having from 1-2 O heteroatomsas ring members. Radical X of formula II is —C(O)— or —S(O)₂—. RadicalR² of formula II is C₁₋₆ alkyl, C₁₋₆ haloalkyl, cycloalkyl having 3-6ring members, C₁₋₆ alkyl-heterocycloalkyl having from 5-6 ring membersand at least 2 N heteroatoms as ring members and substituted with amember benzyl or —C(O)—C₁₋₆ alkyl, phenyl optionally substituted withOH, or C₀₋₆ alkyl-heteroaryl having 5-6 ring members and at least 1 Nheteroatom as a ring member and optionally substituted with halogen.Radical R³ of formula II is H or C₁₋₆ alkyl. Subscript n of formula IIis an integer from 1 to 5. When R¹ is C₁₋₆ haloalkoxy, then R² is C₂₋₆alkyl, C₂₋₆ haloalkyl, cycloalkyl having 3-6 ring members, C₁₋₆alkyl-heterocycloalkyl having from 5-6 ring members and at least 2 Nheteroatoms as ring members and substituted with a member benzyl or—C(O)—C₁₋₆ alkyl, phenyl optionally substituted with OH, or C₀₋₆alkyl-heteroaryl having 5-6 ring members and at least 1 N heteroatom asa ring member and optionally substituted with halogen. The salts andisomers of the compounds of formula II are also encompassed by thepresent invention.

In another aspect, the present invention provides a compound havingformula I:

wherein each R¹ of formula I is H, halogen, C₁₋₆ alkyl, C₁ _(_) ₆alkoxy, C₁ _(_) ₆ haloalkyl, C₁₋₆ haloalkoxy, —O-aryl, heterocycloalkylhaving 5-6 ring members and at least 1 N heteroatom and 1 O heteroatomas ring members, —OH, —NO₂ or —C(O)OR³, wherein at least 1 R¹ is otherthan H. Alternatively, two R¹ groups on adjacent carbons are joined toform a 5-6 membered heterocycloalkyl ring having from 1-2 O heteroatomsas ring members. R² of formula I is C₁₋₆ alkyl and R³ is H or C₁₋₆alkyl. Subscript n of formula I is an integer from 1 to 5. When R¹ offormula I is C₁₋₆ haloalkoxy, then R² is C₂₋₆ alkyl. The salts andisomers of the compounds of formula I are also encompassed by thepresent invention.

In another aspect, the present invention provides a compound havingformula II:

wherein R¹ of formula II is C₁₋₆ haloalkyl or C₁₋₆ haloalkoxy and X is—C(O)— or —S(O)₂—. Radical R² of formula II is C₂₋₆ alkyl, C₂₋₆haloalkyl, cycloalkyl having 3-6 ring members, C₁₋₆alkyl-heterocycloalkyl having from 5-6 ring members and at least 2 Nheteroatoms as ring members and substituted with benzyl or —C(O)—C₁₋₆alkyl, phenyl optionally substituted with OH, or C₀₋₆ alkyl-heteroarylhaving 5-6 ring members and at least 1 N heteroatom as a ring member andoptionally substituted with halogen. The salts and isomers of thecompounds of formula II are also encompassed by the present invention.

In yet another aspect, the present invention provides pharmaceuticalcompositions having a compound of the present invention and apharmaceutically acceptable excipient.

In still another aspect, the present invention provides a method forinhibiting a soluble epoxide hydrolase, the method including contactingthe soluble epoxide hydrolase with an inhibiting amount of a compound ofthe present invention.

In still yet another aspect, the present invention provides a method formonitoring the activity of a soluble epoxide hydrolase, the methodincluding contacting the soluble epoxide hydrolase with an amount of acompound of the present invention sufficient to produce a detectablechange in the fluorescence of the soluble epoxide hydrolase byinteracting with one or more tryptophan residues present in thecatalytic site of said sEH.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative blood pharmacokinetics for AUDA and compounds2, 4, 15, 24 and 25.

FIG. 2 shows blood pharmacokinetics profiles of compounds AUDA, 2, 3, 4,12, 13, 14, 15, 24, 25, 27, and 35.

FIG. 3 shows in vivo exposure (estimated as area under the curve fromFIG. 1, Table 5) as a function of potency (−log IC₅₀) on the homogenous,recombinant human sEH (IC₅₀ from Table 7).

FIG. 4 shows in vivo exposure (estimated as area under the curve fromFIG. 1, Table 5) as a function of potency (−log IC₅₀) on the homogenous,recombinant murine sEH (IC₅₀ from Table 7).

FIG. 5 shows Schemes 1 and 2 for the preparation of the compounds of thepresent invention.

FIG. 6 shows pharmacokinetic data for selected compounds of the presentinvention in non-human primates.

FIG. 7 shows blood pharmacokinetics profiles of compounds 61 (A), 70(B), 69 (C) and 64 (D).

DETAILED DESCRIPTION OF THE INVENTION I. Abbreviations and Definitions

“cis-Epoxyeicosatrienoic acids” (“EETs”) are biomediators synthesized bycytochrome P450 epoxygenases.

“Epoxide hydrolases” (“EH;” EC 3.3.2.3) are enzymes in the alpha/betahydrolase fold family that add water to 3 membered cyclic ethers termedepoxides.

“Soluble epoxide hydrolase” (“sEH”) is an enzyme which in endothelial,smooth muscle and other cell types converts EETs to dihydroxyderivatives called dihydroxyeicosatrienoic acids (“DHETs”). The cloningand sequence of the murine sEH is set forth in Grant et al., J. Biol.Chem. 268(23):17628-17633 (1993). The cloning, sequence, and accessionnumbers of the human sEH sequence are set forth in Beetham et al., Arch.Biochem. Biophys. 305(1):197-201 (1993). The amino acid sequence ofhuman sEH is also set forth as SEQ ID NO:2 of U.S. Pat. No. 5,445,956;the nucleic acid sequence encoding the human sEH is set forth asnucleotides 42-1703 of SEQ ID NO:1 of that patent. The evolution andnomenclature of the gene is discussed in Beetham et al., DNA Cell Biol.14(1):61-71 (1995). Soluble epoxide hydrolase represents a single highlyconserved gene product with over 90% homology between rodent and human(Arand et al., FEBS Lett., 338:251-256 (1994)).

As used herein, the terms “treat”, “treating” and “treatment” refers toany indicia of success in the treatment or amelioration of an injury,pathology, condition, or symptom (e.g., pain), including any objectiveor subjective parameter such as abatement; remission; diminishing ofsymptoms or making the symptom, injury, pathology or condition moretolerable to the patient; decreasing the frequency or duration of thesymptom or condition; or, in some situations, preventing the onset ofthe symptom or condition. The treatment or amelioration of symptoms canbe based on any objective or subjective parameter; including, e.g., theresult of a physical examination.

The term “modulate” refers to the ability of a compound to increase ordecrease the function, or activity, of the associated activity (e.g.,soluble epoxide hydrolase). “Modulation”, as used herein in its variousforms, is meant to include antagonism and partial antagonism of theactivity associated with sEH. Inhibitors of sEH are compounds that,e.g., bind to, partially or totally block the enzyme's activity.

The term “compound” as used herein is intended to encompass not only thespecified molecular entity but also its pharmaceutically acceptable,pharmacologically active derivatives, including, but not limited to,salts, prodrug conjugates such as esters and amides, metabolites,hydrates, solvates and the like.

The term “composition” as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts. By“pharmaceutically acceptable” it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The “subject” is defined herein to include animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. In someembodiments, the subject is a human.

As used herein, the term “sEH-mediated disease or condition” and thelike refers to a disease or condition characterized by less than orgreater than normal, sEH activity. A sEH-mediated disease or conditionis one in which modulation of sEH results in some effect on theunderlying condition or disease (e.g., a sEH inhibitor or antagonistresults in some improvement in patient well-being in at least somepatients).

“Parenchyma” refers to the tissue characteristic of an organ, asdistinguished from associated connective or supporting tissues.

“Chronic Obstructive Pulmonary Disease” or “COPD” is also sometimesknown as “chronic obstructive airway disease”, “chronic obstructive lungdisease”, and “chronic airways disease.” COPD is generally defined as adisorder characterized by reduced maximal expiratory flow and slowforced emptying of the lungs. COPD is considered to encompass tworelated conditions, emphysema and chronic bronchitis. COPD can bediagnosed by the general practitioner using art recognized techniques,such as the patient's forced vital capacity (“FVC”), the maximum volumeof air that can be forcibly expelled after a maximal inhalation. In theoffices of general practitioners, the FVC is typically approximated by a6 second maximal exhalation through a spirometer. The definition,diagnosis and treatment of COPD, emphysema, and chronic bronchitis arewell known in the art and discussed in detail by, for example, Honig andIngram, in Harrison's Principles of Internal Medicine (Fauci et al.,eds.), 14th Ed., 1998, McGraw-Hill, New York, pp. 1451-1460 (hereafter,“Harrison's Principles of Internal Medicine”).

“Emphysema” is a disease of the lungs characterized by permanentdestructive enlargement of the airspaces distal to the terminalbronchioles without obvious fibrosis.

“Chronic bronchitis” is a disease of the lungs characterized by chronicbronchial secretions which last for most days of a month, for threemonths a year, for two years.

As the names imply, “obstructive pulmonary disease” and “obstructivelung disease” refer to obstructive diseases, as opposed to restrictivediseases. These diseases particularly include COPD, bronchial asthma andsmall airway disease.

“Small airway disease.” There is a distinct minority of patients whoseairflow obstruction is due, solely or predominantly to involvement ofthe small airways. These are defined as airways less than 2 mm indiameter and correspond to small cartilaginous bronchi, terminalbronchioles and respiratory bronchioles. Small airway disease (SAD)represents luminal obstruction by inflammatory and fibrotic changes thatincrease airway resistance. The obstruction may be transient orpermanent.

The “interstitial lung diseases (ILDs)” are a group of conditionsinvolving the alveolar walls, perialveolar tissues, and contiguoussupporting structures. As discussed on the website of the American LungAssociation, the tissue between the air sacs of the lung is theinterstitium, and this is the tissue affected by fibrosis in thedisease. Persons with the disease have difficulty breathing in becauseof the stiffness of the lung tissue but, in contrast to persons withobstructive lung disease, have no difficulty breathing out. Thedefinition, diagnosis and treatment of interstitial lung diseases arewell known in the art and discussed in detail by, for example, Reynolds,H. Y., in Harrison's Principles of Internal Medicine, supra, at pp.1460-1466. Reynolds notes that, while ILDs have various initiatingevents, the immunopathological responses of lung tissue are limited andthe ILDs therefore have common features.

“Idiopathic pulmonary fibrosis,” or “IPF,” is considered the prototypeILD. Although it is idiopathic in that the cause is not known, Reynolds,supra, notes that the term refers to a well-defined clinical entity.

“Bronchoalveolar lavage,” or “BAL,” is a test which permits removal andexamination of cells from the lower respiratory tract and is used inhumans as a diagnostic procedure for pulmonary disorders such as IPF. Inhuman patients, it is usually performed during bronchoscopy.

“Inhibition”, “inhibits”, “inhibiting” and “inhibitor” refer to acompound that prohibits or a method of prohibiting, a specific action orfunction.

As used herein, the term “contacting” refers to the process of bringinginto contact at least two distinct species such that they can react. Itshould be appreciated, however, the resulting reaction product can beproduced directly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture.

As used herein, the term “alkyl” refers to a saturated hydrocarbonradical which may be straight-chain or branched-chain (for example,ethyl, isopropyl, t-amyl, or 2,5-dimethylhexyl). This definition appliesboth when the term is used alone and when it is used as part of acompound term, such as “arylalkyl,” “alkylamino,” “alkylheteroaryl,”“alkylheterocycloalkyl” and similar terms. In some embodiments, alkylgroups are those containing 1 to 24 carbon atoms. All numerical rangesin this specification and claims are intended to be inclusive of theirupper and lower limits. Additionally, the alkyl and heteroalkyl groupsmay be attached to other moieties at any position on the alkyl orheteroalkyl radical which would otherwise be occupied by a hydrogen atom(such as, for example, 2-pentyl, 2-methylpent-1-yl and 2-propyloxy).Divalent alkyl groups may be referred to as “alkylene,” and divalentheteroalkyl groups may be referred to as “heteroalkylene”. The alkyl,alkylene, and heteroalkylene moieties may also be optionally substitutedwith halogen atoms, or other groups such as oxo, cyano, nitro, alkyl,alkylamino, carboxyl, hydroxyl, alkoxy, aryloxy, and the like.

The terms “cycloalkyl” and “cycloalkylene” refer to a saturatedhydrocarbon ring and includes bicyclic and polycyclic rings. Similarly,cycloalkyl and cycloalkylene groups having a heteroatom (e.g., N, O orS) in place of a carbon ring atom may be referred to as“heterocycloalkyl” and “heterocycloalkylene,” respectively. Examples ofcycloalkyl and heterocycloalkyl groups are, for example, cyclohexyl,norbomyl, adamantyl, morpholinyl, thiomorpholinyl, dioxothiomorpholinyl,and the like. The cycloalkyl and heterocycloalkyl moieties may also beoptionally substituted with halogen atoms, or other groups such asnitro, alkyl, alkylamino, carboxyl, alkoxy, aryloxy and the like. Insome embodiments, cycloalkyl and cycloalkylene moieties are those having3 to 12 carbon atoms in the ring (e.g., cyclohexyl, cyclooctyl,norbomyl, adamantyl, and the like). In some embodiments,heterocycloalkyl and heterocycloalkylene moieties are those having 1 to3 hetero atoms in the ring (e.g., morpholinyl, thiomorpholinyl,dioxothiomorpholinyl, piperidinyl and the like). Additionally, the term“(cycloalkyl)alkyr” refers to a group having a cycloalkyl moietyattached to an alkyl moiety. Examples are cyclohexylmethyl,cyclohexylethyl and cyclopentylpropyl.

The term “alkenyl” as used herein refers to an alkyl group as describedabove which contains one or more sites of unsaturation that is a doublebond. Similarly, the term “alkynyl” as used herein refers to an alkylgroup as described above which contains one or more sites ofunsaturation that is a triple bond.

The term “alkoxy” refers to an alkyl radical as described above whichalso bears an oxygen substituent which is capable of covalent attachmentto another hydrocarbon radical (such as, for example, methoxy, ethoxyand t-butoxy).

The term “aryl” refers to an aromatic carbocyclic substituent which maybe a single ring or multiple rings which are fused together, linkedcovalently or linked to a common group such as an ethylene or methylenemoiety. Similarly, aryl groups having a heteroatom (e.g., N, O or S) inplace of a carbon ring atom are referred to as “heteroaryl”. Examples ofaryl and heteroaryl groups are, for example, phenyl, naphthyl, biphenyl,diphenylmethyl, thienyl, pyridyl and quinoxalyl. The aryl and heteroarylmoieties may also be optionally substituted with halogen atoms, or othergroups such as nitro, alkyl, alkylamino, carboxyl, alkoxy, phenoxy andthe like. Additionally, the aryl and heteroaryl groups may be attachedto other moieties at any position on the aryl or heteroaryl radicalwhich would otherwise be occupied by a hydrogen atom (such as, forexample, 2-pyridyl, 3-pyridyl and 4-pyridyl). Divalent aryl groups are“arylene”, and divalent heteroaryl groups are referred to as“heteroarylene” such as those groups used as linkers in the presentinvention.

The terms “arylalkyl” and “alkylaryl”, “refer to an aryl radicalattached directly to an alkyl group. Likewise, the terms “arylalkenyl”and “aryloxyalkyl” refer to an alkenyl group, or an oxygen which isattached to an alkyl group, respectively. For brevity, aryl as part of acombined term as above, is meant to include heteroaryl as well. The term“aryloxy” refers to an aryl radical as described above which also bearsan oxygen substituent which is capable of covalent attachment to anotherradical (such as, for example, phenoxy, naphthyloxy, and pyridyloxy).

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” and“haloalkoxy” are meant to include monohaloalkyl(oxy) andpolyhaloalkyl(oxy). For example, the term “C₁-C₆ haloalkyl” is mean toinclude trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “hetero” as used in a “heteroatom-containing alkyl group” (a“heteroalkyl” group) or a “heteroatom-containing aryl group” (a“heteroaryl” group) refers to a molecule, linkage or substituent inwhich one or more carbon atoms are replaced with an atom other thancarbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typicallynitrogen, oxygen or sulfur or more than one non-carbon atom (e.g.,sulfonamide). Similarly, the term “heteroalkyl” refers to an alkylsubstituent that is heteroatom-containing, the terms “heterocyclic”,“heterocycle”, “heterocycloalkyl” or “heterocyclyl” refer to a cyclicsubstituent or group that is heteroatom-containing and is eitheraromatic or non-aromatic. The terms “heteroaryl” and “heteroaromatic”respectively refer to “aryl” and “aromatic” substituents that areheteroatom-containing, and the like. The terms “heterocyclic” and“heterocyclyl” include the terms “heteroaryl” and “heteroaromatic”. Insome embodiments, heterocyclic moieties are those having 1 to 3 heteroatoms in the ring. Examples of heteroalkyl groups include alkoxy,alkoxyaryl, alkylsulfanyl-substituted alkyl, N-alkylated amino alkyl,and the like. Examples of heteroaryl substituents include pyrrolyl,pyrrolidinyl, pyridinyl, quinolinyl, indolyl, pyrimidinyl, imidazolyl,1,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatom-containingcyclic nonaromatic groups are morpholinyl, piperazinyl, piperidinyl,etc.

The term “carboxylic acid analog” refers to a variety of groups havingan acidic moiety that are capable of mimicking a carboxylic acidresidue. Examples of such groups are sulfonic acids, sulfinic acids,phosphoric acids, phosphonic acids, phosphinic acids, sulfonamides, andheterocyclic moieties such as, for example, imidazoles, triazoles andtetrazoles.

The term “substituted” refers to the replacement of an atom or a groupof atoms of a compound with another atom or group of atoms. For example,an atom or a group of atoms may be substituted with one or more of thefollowing substituents or groups: halo, nitro, C₁-C₈alkyl,C₁-C₈alkylamino, hydroxyC₁-C₈alkyl, haloC₁-C₈alkyl, carboxyl, hydroxyl,C₁-C₈alkoxy, C₁-C₈alkoxyC₁-C₈alkoxy, thioC₁-C₈alkyl, aryl, aryloxy,C₃-C₈cycloalkyl, C₃-C₈cycloalkylC₁-C₈alkyl, heteroaryl, arylC₁-C₈alkyl,heteroarylC₁-C₈alkyl, C₂-C₈alkenyl containing 1 to 2 double bonds,C₂-C₈alkynyl containing 1 to 2 triple bonds, C₄-C₈alk(en)(yn)yl groups,cyano, formyl, C₁-C₈alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl,C₁-C₈alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl,C₁-C₈alkylaminocarbonyl, C₁-C₈dialkylaminocarbonyl, arylaminocarbonyl,diarylaminocarbonyl, arylC₁-C₈alkylaminocarbonyl, haloC₁-C₈alkoxy,C₂-C₈alkenyloxy, C₂-C₈alkynyloxy, arylC₁-C₈alkoxy, aminoC₁-C₈alkyl,C₁-C₈alkylaminoC₁-C₈alkyl, C₁-C₈dialkylaminoC₁-C₈alkyl,arylaminoC₁-C₈alkyl, amino, C₁-C₈dialkylamino, arylamino,arylC₁-C₈alkylamino, C₁-C₈alkylcarbonylamino, arylcarbonylamino, azido,mercapto, C₁-C₈alkylthio, arylthio, haloC₁-C₈alkylthio, thiocyano,isothiocyano, C₁-C₈alkylsulfinyl, C₁-C₈alkylsulfonyl, arylsulfinyl,arylsulfonyl, aminosulfonyl, C₁-C₈alkylaminosulfonyl,C₁-C₈dialkylaminosulfonyl and arylaminosulfonyl. When the term“substituted” appears prior to a list of possible substituted groups, itis intended that the term apply to every member of that group.

The term “unsubstituted” refers to a native compound that lacksreplacement of an atom or a group of atoms.

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods of the present invention. Illustrativeexamples of pharmaceutically acceptable salts are mineral acid(hydrochloric acid, hydrobromic acid, phosphoric acid, and the like)salts, organic acid (acetic acid, propionic acid, glutamic acid, citricacid and the like) salts, quaternary ammonium (methyl iodide, ethyliodide, and the like) salts. It is understood that the pharmaceuticallyacceptable salts are non-toxic. Additional information on suitablepharmaceutically acceptable salts can be found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, which is incorporated herein by reference.

As used herein, the term “pharmaceutically acceptable excipient” refersto a substance that aids the administration of an active agent to andabsorption by a subject. Pharmaceutical excipients useful in the presentinvention include, but are not limited to, binders, fillers,disintegrants, lubricants, coatings, sweeteners, flavors and colors. Oneof skill in the art will recognize that other pharmaceutical excipientsare useful in the present invention.

As used herein, the terms “therapeutically effective amount or dose” or“therapeutically sufficient amount or dose” or “effective or sufficientamount or dose” refer to a dose that produces therapeutic effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, thetherapeutically effective dose can often be lower than the conventionaltherapeutically effective dose for non-sensitized cells.

II. General

The present invention derives from the discovery that 1,3-disubstitutedureas (or the corresponding amides or carbamates, also referred to asthe primary pharmacophore) can be further functionalized to provide morepotent sEH inhibitors with improved physical properties. As describedherein, the introduction of a heterocyclic moiety can increase watersolubility and oral availability of sEH inhibitors (see below). Thecombination of these moieties provides a variety of compounds ofincreased water solubility.

The discovery of the heterocyclic pharmacophores has also led to theemployment of combinatorial chemistry approaches for establishing a widespectrum of compounds having sEH inhibitory activity. The polarpharmacophores divide the molecule into domains each of which can beeasily manipulated by common chemical approaches in a combinatorialmanner, leading to the design and confirmation of novel orally availabletherapeutic agents for the treatment of diseases such as hypertensionand vascular inflammation. The agents of the present invention treatsuch diseases while simultaneously increasing sodium excretion, reducingvascular and renal inflammation, and reducing male erectile dysfunction.As shown below (see Examples and Figures), alterations in solubility,bioavailability and pharmacological properties leads to compounds thatcan alter the regulatory lipids of experimental animals increasing therelative amounts of epoxy arachidonate derivatives when compared eitherto their diol products or to the proinflammatory and hypertensivehydroxyeicosatetraenoic acids (HETEs). Since epoxy arachidonates areanti-hypertensive and anti-inflammatory, altering the lipid ratios canlead to reduced blood pressure and reduced vascular and renalinflammation. This approach has been validated as reported in U.S.patent application Ser. Nos. 10/817,334 and 11/256,685 which are hereinincorporated by reference in their entirety.

The heterocyclic group improves water solubility of sEH inhibitors aswell as the specificity for the sEH, and a wide diversity offunctionalities such as an ester, amide, carbamate, or similarfunctionalities capable of donating or accepting a hydrogen bondsimilarly can contribute to this polar group. For example, inpharmaceutical chemistry heterocyclic groups are commonly used to mimiccarbonyls as hydrogen bond donors and acceptors. Of course the primary,secondary and tertiary pharmacophore groups can be combined in a singlemolecule with suitable spacers to improve activity or present theinhibitor as a prodrug.

III. Compounds for Inhibiting Soluble Epoxide Hydrolases

In addition to the methods provided below, the present inventionprovides compounds that can inhibit the activity of soluble epoxidehydrolases. In particular, the present invention provides compoundshaving a formula selected from the formulas below. The compounds of thepresent invention have chemical handles that allow attachment offluorescent molecules useful for binding studies such as fluorescencepolarization or FRET. Affinity ligands can also be attached to thecompounds of the present invention.

In some embodiments, the present invention provides a compound havingformula II:

wherein each R¹ of formula II is independently H, halogen, C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, —O-aryl, heterocycloalkylhaving 5-6 ring members and at least 1 N heteroatom and 1 O heteroatomas ring members, —OH, —NO₂ or —C(O)OR³, wherein at least 1 R¹ is otherthan H. Alternatively, two R¹ groups on adjacent carbons are joined toform a 5-6 membered heterocycloalkyl ring having from 1-2 O heteroatomsas ring members. Radical X of formula II is —C(O)— or —S(O)₂—. RadicalR² of formula II is C₁₋₆ alkyl, C₁₋₆ haloalkyl, cycloalkyl having 3-6ring members, C₁₋₆ alkyl-heterocycloalkyl having from 5-6 ring membersand at least 2 N heteroatoms as ring members and substituted with amember benzyl or —C(O)—C₁₋₆ alkyl, phenyl optionally substituted withOH, or C₀₋₆ alkyl-heteroaryl having 5-6 ring members and at least 1 Nheteroatom as a ring member and optionally substituted with halogen.Radical R³ of formula II is H or C₁₋₆ alkyl. Subscript n of formula IIis an integer from 1 to 5. When R¹ is C₁₋₆ haloalkoxy, then R² is C₂₋₆alkyl, C₂₋₆ haloalkyl, cycloalkyl having 3-6 ring members, C₁₋₆alkyl-heterocycloalkyl having from 5-6 ring members and at least 2 Nheteroatoms as ring members and substituted with a member benzyl or—C(O)—C₁₋₆ alkyl, phenyl optionally substituted with OH, or C₀₋₆alkyl-heteroaryl having 5-6 ring members and at least 1 N heteroatom asa ring member and optionally substituted with halogen. The salts andisomers of the compounds of formula II are also encompassed by thepresent invention.

In other embodiments, when R¹ is C₁₋₆ haloalkoxy and X is —C(O)—, thenR² is cycloalkyl having 3-6 ring members, C₁₋₆ alkyl-heterocycloalkylhaving from 5-6 ring members and at least 2 N heteroatoms as ringmembers and substituted with a member benzyl or —C(O)—C₁₋₆ alkyl, phenyloptionally substituted with OH, or C₀₋₆ alkyl-heteroaryl having 5-6 ringmembers and at least 1 N heteroatom as a ring member and optionallysubstituted with halogen. In some other embodiments, when R¹ is C₁₋₆haloalkoxy and X is —S(O)2-, then R² is C₂₋₆ haloalkyl, cycloalkylhaving 3-6 ring members, C₁₋₆ alkyl-heterocycloalkyl having from 5-6ring members and at least 2 N heteroatoms as ring members andsubstituted with a member benzyl or —C(O)—C₁₋₆ alkyl, phenyl optionallysubstituted with OH, or C₀₋₆ alkyl-heteroaryl having 5-6 ring membersand at least 1 N heteroatom as a ring member and optionally substitutedwith halogen.

In some embodiments, the present invention provides a compound havingformula I:

wherein each R¹ of formula I is H, halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, —O-aryl, heterocycloalkyl having 5-6ring members and at least 1 N heteroatom and 1 O heteroatom as ringmembers, —OH, —NO2 or —C(O)OR³, wherein at least 1 R¹ is other than H.Alternatively, two R¹ groups on adjacent carbons are joined to form a5-6 membered heterocycloalkyl ring having from 1-2 O heteroatoms as ringmembers. R² of formula I is C₁₋₆ alkyl and R³ is H or C₁₋₆ alkyl.Subscript n of formula I is an integer from 1 to 5. When R¹ of formula Iis C₁₋₆ haloalkoxy, then R² is C₂₋₆ alkyl. The salts and isomers thecompounds of formula I are also encompassed by the present invention.

In other embodiments, the compounds of the present invention are thosehaving formula Ia:

wherein R^(1a), R^(1b) and R^(1c) are each independently selected fromthe members of the group defined by R¹. In some other embodiments, thecompounds of the present invention include those having the formula Ib:

In another embodiment, each R¹ of formula I is halogen, C₁₋₆ alkyl, C₁₋₆alkoxy, —O-aryl, heterocycloalkyl having 5-6 ring members and at least 1N heteroatom and 1 O heteroatom as ring members, —OH, —NO₂ or —C(O)OR³.In other embodiments, each R¹ of formula I is halogen or C₁₋₆ haloalkyl.In some other embodiments, each R¹ of formula I is halogen, C₁₋₆haloalkyl or —O-aryl. Alternatively, two R¹ groups on adjacent carbonsare joined to form a 5 membered heterocycloalkyl ring having 2 Oheteroatoms as ring members. In still other embodiments, each R¹ offormula I is Cl, perfluoro-isopropyl or phenoxy. In yet otherembodiments, R² of formula I is ethyl.

In some embodiments, the compounds of formula I have the followingformula:

In other embodiments, the present invention provides a compound havingformula II:

wherein R¹ of formula II is C₁₋₆ haloalkyl or C₁₋₆ haloalkoxy and X is—C(O)— or —S(O)2-. Radical R² of formula II is C₂₋₆ alkyl, C₂₋₆haloalkyl, cycloalkyl having 3-6 ring members, C₁₋₆alkyl-heterocycloalkyl having from 5-6 ring members and at least 2 Nheteroatoms as ring members and substituted with a member the groupconsisting of benzyl and —C(O)—C₁₋₆ alkyl, phenyl optionally substitutedwith OH, or C₀₋₆ alkyl-heteroaryl having 5-6 ring members and at least 1N heteroatom as a ring member and optionally substituted with halogen.The salts and isomers of the compounds of formula II are alsoencompassed by the present invention.

In another embodiments, the present invention provides a compound havingformula II, wherein R¹ of formula II is C₁₋₆ haloalkoxy and X is —C(O)—or —S(O)2-. Radical R² of formula II is C₂₋₆ alkyl, C₂₋₆ haloalkyl,cycloalkyl having 3-6 ring members, C₁₋₆ alkyl-heterocycloalkyl havingfrom 5-6 ring members and at least 2 N heteroatoms as ring members andsubstituted with a member the group consisting of benzyl and —C(O)—C₁₋₆alkyl, phenyl optionally substituted with OH, or C₀₋₆ alkyl-heteroarylhaving 5-6 ring members and at least 1 N heteroatom as a ring member andoptionally substituted with halogen. The salts and isomers of thecompounds of formula II are also encompassed by the present invention.

In some other embodiments, the compounds of the present inventioninclude those having formula IIa:

In some other embodiments, the compounds of the present inventioninclude those having formula IIb:

wherein R² of formula lib is C₂₋₆ haloalkyl, cycloalkyl having 3-6 ringmembers, C₁₋₆ alkyl-heterocycloalkyl having from 5-6 ring members and atleast 2 N heteroatoms as ring members and substituted with a member thegroup consisting of benzyl and —C(O)—C₁₋₆ alkyl, phenyl substituted withOH, or C₀₋₆ alkyl-heteroaryl having 5-6 ring members and at least 1 Nheteroatom as a ring member and optionally substituted with a halogen.

In yet other embodiments, R² of formula I is —CH2-heterocycloalkylhaving 6 ring members and 2 N heteroatoms as ring members andsubstituted with acetyl. In still yet other embodiments, the compoundhas the formula:

In another embodiment, the compounds of the present invention are thosehaving formula IIc:

wherein R² of formula IIc is C₂₋₆ alkyl or unsubstituted phenyl. In someembodiments, the compound of formula lie has the formula:

In another embodiment, the compounds of the present invention are thosehaving formula IIe:

In another embodiment, the compounds of the present invention are thosehaving formula IIe:

In another embodiment, the compounds of the present invention are thosehaving formula IIf:

In another embodiment, the compounds of the present invention are:

In another embodiment, the compounds of the present invention are:

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention.

Pharmaceutically acceptable salts of the acidic compounds of the presentinvention are salts formed with bases, namely cationic salts such asalkali and alkaline earth metal salts, such as sodium, lithium,potassium, calcium, magnesium, as well as ammonium salts, such asammonium, trimethyl-ammonium, diethylammonium, andtris-(hydroxymethyl)-methyl-ammonium salts.

Similarly acid addition salts, such as of mineral acids, organiccarboxylic and organic sulfonic acids, e.g., hydrochloric acid,methanesulfonic acid, maleic acid, are also possible provided a basicgroup, such as pyridyl, constitutes part of the structure.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In some embodiments, sEH inhibitors for treating hypertension or highblood pressure have an IC₅₀ in a defined assay of less than 50 μM. Inanother embodiment, the compounds have an IC₅₀ of 1 μM or less. Inanother embodiment, the compounds have an IC₅₀ of 500 nM or less. Inanother embodiment, the compounds have an IC₅₀ of 150 nM or less. Inanother embodiment, the compounds have an IC₅₀ of 100 nM or less. Inanother embodiment, the compounds have an IC₅₀ of 50 nM or less. Inanother embodiment, the compounds have an IC₅₀ of 1 nM or less.

The compounds of the present invention can be prepared by a variety ofmethods as outlined generally in the schemes. It should be noted thatthe synthetic conditions illustrated in the following scheme are alsoapplicable to those inhibitors based on 4-aminomethylpiperidine (thosewith a CH₂ spacer).

Scheme 1 outlines the two general synthetic routes used to form theunsymmetrical 1,3-disubstituted urea pharmacophore. Aryl isocyanateswere purchased or formed from their corresponding anilines by reactionwith triphosgene in the presence of aqueous base (J. Org. Chem. 1996,61, 3929-3934). The heptafluoroisopropylanilines required for compounds38 and 39 were prepared as described (EP 1006102, Jun. 7, 2000).

Amine 1 was prepared from 4-aminopiperidine by protection of the primaryamine as its benzyl imine (Bioorg. Med. Chem. 2003, 11, 4225-4234),reaction with propionyl chloride in the presence of triethylamine andsubsequent deprotection. All isocyanates were reacted with amine 1 togive the desired (1-propionylpiperidin-4-yl)ureas 2-16, 18-21 and 24-40.Saponification of methyl ester 21 with methanolic NaOH afforded benzoicacid 22. Phenol 23 was prepared via 4-benzyloxyisocyanate to avoidformation of a carbamate side product.

Compounds 17 and 31 were prepared by conversion of the correspondinganiline to the intermediate 4-nitrophenyl carbamate, which was thenreacted with amine 1 to give the desired urea.

Intermediate 41 (Scheme 2) was prepared by the reaction of4-trifluoromethoxyphenyl isocyanate with 1-BOC-4-aminopiperidine. BOCde-protection gave piperidine 42, which was converted to N-acylcompounds 47-50 and 52 by an EDCI mediated coupling reaction with therespective carboxylic acid (Bioorg. Med. Chem. 2003, 11, 4225-4234).Acetylpiperazine 51 was prepared by de-benzylation of 50 and subsequentN-acetylation. Trifluoroacetyl compound 53 was prepared by the reactionof intermediate 42 with ethyl trifluoroacetate. Trihydroxybenzoylcompound 52 was prepared by coupling of 42 with tris O-benzyl protectedgallic acid (44) followed by hydrogenolysis (J. Med. Chem. 2006, 49,2829-2837). Intermediate 42 was also converted to N-sulfonyl compounds55-59 by reaction with the respective sulfonyl chlorides.

IV. Pharmaceutical Compositions

In another embodiment, the present invention provides a pharmaceuticalcomposition, including a compound of the present invention and apharmaceutically acceptable excipient.

The compounds of the present invention can be prepared and administeredin a wide variety of oral, parenteral and topical dosage forms. Oralpreparations include tablets, pills, powder, dragees, capsules, liquids,lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitablefor ingestion by the patient. The compounds of the present invention canalso be administered by injection, that is, intravenously,intramuscularly, intracutaneously, subcutaneously, intraduodenally, orintraperitoneally. Also, the compounds described herein can beadministered by inhalation, for example, intranasally. Additionally, thecompounds of the present invention can be administered transdermally.The compounds of the present invention can also be administered byintraocular, intravaginal, and intrarectal routes includingsuppositories, insufflation, powders and aerosol formulations (forexamples of steroid inhalants, see Rohatagi, J. Clin. Pharmacol.35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111,1995). Accordingly, the present invention also provides pharmaceuticalcompositions including a pharmaceutically acceptable carrier orexcipient and either a compound of the present invention, or apharmaceutically acceptable salt of a compound of the present invention.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Asolid carrier can be one or more substances, which may also act asdiluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.(“Remington's”).

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain from 5% or 10% to70% of the active compound.

The compositions typically include a conventional pharmaceutical carrieror excipient and may additionally include other medicinal agents,carriers, adjuvants, diluents, tissue permeation enhancers,solubilizers, and the like. Preferably, the composition will containabout 0.01% to about 90%, preferably about 0.1% to about 75%, morepreferably about 0.1% to 50%), still more preferably about 0.1% to 10%by weight of a ligand of the present invention or a combination thereof,with the remainder consisting of suitable pharmaceutical carrier and/orexcipients. Appropriate excipients can be tailored to the particularcomposition and route of administration by methods well known in theart, e.g., Remington's Pharmaceutical Sciences, supra.

Suitable solid excipients include, but are not limited to, magnesiumcarbonate; magnesium stearate; calcium phosphate; calcium silicate;talc; pectin; dextran, dextrin, and cyclodextrin inclusion complexes; alow melting wax; cocoa butter; carbohydrates; sugars including, but notlimited to, lactose, dextrose, sucrose, mannitol, or sorbitol; starchesincluding, but not limited to, starch from corn, wheat, rice, potato, orother plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic, tragacanth, and acacia; as well as proteinsincluding, but not limited to, gelatin, collagen; microcrystallinecellulose, water, saline, syrup, ethylcellulose, and polyacrylic acidssuch as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc.;lubricating agents; mineral oil; wetting agents; emulsifying agents;suspending agents; preserving agents such as methyl-, ethyl-, andpropyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents suchas inorganic and organic acids and bases; sweetening agents; andflavoring agents; biodegradable polymer beads. If desired,disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, alginates, or asalt thereof, such as sodium alginate.

A pharmaceutically acceptable carrier may include physiologicallyacceptable compounds that act, for example, to stabilize the compoundsof the present invention or modulate their absorption, or otherexcipients as desired. Physiologically acceptable compounds include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients. Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the route of administration of the compounds ofthe present invention and on the particular physio-chemicalcharacteristics of the compounds of the present invention.

Generally, such carriers should be nontoxic to recipients at the dosagesand concentrations employed. Ordinarily, the preparation of suchcompositions entails combining the therapeutic agent with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, maltose, sucrose or dextrins, chelating agents suchas EDTA, glutathione and other stabilizers and excipients. Neutralbuffered saline or saline mixed with nonspecific serum albumin areexemplary appropriate diluents.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical preparations of theinvention can also be used orally using, for example, push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and acoating such as glycerol or sorbitol. Push-fit capsules can containcompounds of the present invention mixed with a filler or binders suchas lactose or starches, lubricants such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the compounds of thepresent invention may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycol withor without stabilizers.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Oil suspensions can be formulated by suspending a compound of thepresent invention in a vegetable oil, such as arachis oil, olive oil,sesame oil or coconut oil, or in a mineral oil such as liquid paraffin;or a mixture of these. The oil suspensions can contain a thickeningagent, such as beeswax, hard paraffin or cetyl alcohol. Sweeteningagents can be added to provide a palatable oral preparation, such asglycerol, sorbitol or sucrose. These formulations can be preserved bythe addition of an antioxidant such as ascorbic acid. As an example ofan injectable oil vehicle, see Minto, J. Pharmacol. Exp. Ther.281:93-102, 1997. The pharmaceutical formulations of the invention canalso be in the form of oil-in-water emulsions. The oily phase can be avegetable oil or a mineral oil, described above, or a mixture of these.Suitable emulsifying agents include naturally-occurring gums, such asgum acacia and gum tragacanth, naturally occurring phosphatides, such assoybean lecithin, esters or partial esters derived from fatty acids andhexitol anhydrides, such as sorbitan mono-oleate, and condensationproducts of these partial esters with ethylene oxide, such aspolyoxyethylene sorbitan mono-oleate. The emulsion can also containsweetening agents and flavoring agents, as in the formulation of syrupsand elixirs. Such formulations can also contain a demulcent, apreservative, or a coloring agent.

V. Administration

Administration of the compounds of the present invention with a suitablepharmaceutical excipient as necessary can be carried out via any of theaccepted modes of administration. Thus, administration can be, forexample, intravenous, topical, subcutaneous, transcutaneous,transdermal, intramuscular, oral, intra joint, parenteral,intra-arteriole, intradermal, intraventricular, intracranial,intraperitoneal, intralesional, intranasal, rectal, vaginal, or byinhalation. Administration may also be directly to the bone surfaceand/or into tissues surrounding the bone.

The compositions containing a compound or a combination of compounds ofthe present invention may be administered repeatedly, e.g., at least 2,3, 4, 5, 6, 7, 8, or more times, or the composition may be administeredby continuous infusion. Suitable sites of administration include, butare not limited to, skin, bronchial, gastrointestinal, anal, vaginal,eye, and ear. The formulations may take the form of solid, semi-solid,lyophilized powder, or liquid dosage forms, such as, for example,tablets, pills, capsules, powders, solutions, suspensions, emulsions,suppositories, retention enemas, creams, ointments, lotions, gels,aerosols, or the like, preferably in unit dosage forms suitable forsimple administration of precise dosages.

The pharmaceutical preparations are typically delivered to a mammal,including humans and non-human mammals. Non-human mammals treated usingthe present methods include domesticated animals (i.e., canine, feline,murine, rodentia, and lagomorpha) and agricultural animals (bovine,equine, ovine, porcine).

The pharmaceutical preparation is preferably in unit dosage form. Theterm “unit dosage form” refers to physically discrete units suitable asunitary dosages for human subjects and other mammals (e.g., dogs), eachunit containing a predetermined quantity of active material calculatedto produce the desired onset, tolerability, and/or therapeutic effects,in association with a suitable pharmaceutical excipient (e.g., anampoule). In addition, more concentrated compositions may be prepared,from which the more dilute unit dosage compositions may then beproduced. The more concentrated compositions thus will containsubstantially more than, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more times the amount of a compound or a combination of compounds. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packaged tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form. The composition can, if desired, also contain othercompatible therapeutic agents. Preferred pharmaceutical preparations candeliver the compounds of the invention in a sustained releaseformulation.

Methods for preparing such dosage forms are known to those skilled inthe art (see, for example, Remington's Pharmaceutical Sciences, 18thEd., Mack Publishing Co., Easton, Pa. (1990)). The composition to beadministered contains a quantity of the compound or combination ofcompounds in a pharmaceutically effective amount for relief of acondition being treated (e.g., osteoporosis) when administered inaccordance with the teachings of this invention. In addition,pharmaceutically acceptable salts of the compounds of the presentinvention (e.g., acid addition salts) may be prepared and included inthe compositions using standard procedures known to those skilled in theart of synthetic organic chemistry and described, e.g., by J. March,Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed.(New York: Wiley-Interscience, 1992).

For oral administration, the compositions can be in the form of tablets,capsules, emulsions, suspensions, solutions, syrups, sprays, lozenges,powders, and sustained-release formulations. Suitable excipients fororal administration include pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose,glucose, gelatin, sucrose, magnesium carbonate, and the like.

In some embodiments, the pharmaceutical compositions take the form of apill, tablet, or capsule, and thus, the composition can contain, alongwith the compounds or combination of compounds, any of the following: adiluent such as lactose, sucrose, dicalcium phosphate, and the like; adisintegrant such as starch or derivatives thereof; a lubricant such asmagnesium stearate and the like; and a binder such a starch, gum acacia,polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof. Thecompounds can also be formulated into a suppository disposed, forexample, in a polyethylene glycol (PEG) carrier.

Liquid compositions can be prepared by dissolving or dispersing acompound or a combination of compounds and optionally one or morepharmaceutically acceptable adjuvants in a carrier such as, for example,aqueous saline (e.g., 0.9% w/v sodium chloride), aqueous dextrose,glycerol, ethanol, and the like, to form a solution or suspension, e.g.,for oral, topical, or intravenous administration. The compounds of thepresent invention can also be formulated into a retention enema.

For topical administration, the compositions of the present inventioncan be in the form of emulsions, lotions, gels, creams, jellies,solutions, suspensions, ointments, and transdermal patches. For deliveryby inhalation, the composition can be delivered as a dry powder or inliquid form via a nebulizer. For parenteral administration, thecompositions can be in the form of sterile injectable solutions andsterile packaged powders. Preferably, injectable solutions areformulated at a pH of about 4.5 to about 7.5.

The compositions of the present invention can also be provided in alyophilized form. Such compositions may include a buffer, e.g.,bicarbonate, for reconstitution prior to administration, or the buffermay be included in the lyophilized composition for reconstitution with,e.g., water. The lyophilized composition may further comprise a suitablevasoconstrictor, e.g., epinephrine. The lyophilized composition can beprovided in a syringe, optionally packaged in combination with thebuffer for reconstitution, such that the reconstituted composition canbe immediately administered to a patient.

Generally, administered dosages will be effective to deliver picomolarto micromolar concentrations of the compound to the appropriate site orsites. However, one of ordinary skill in the art understands that thedose administered will vary depending on a number of factors, including,but not limited to, the particular compound or set of compounds to beadministered, the mode of administration, the type of application (e.g.,imaging, therapeutic), the age of the patient, and the physicalcondition of the patient. Preferably, the smallest dose andconcentration required to produce the desired result should be used.Dosage should be appropriately adjusted for children, the elderly,debilitated patients, and patients with cardiac and/or liver disease.Further guidance can be obtained from studies known in the art usingexperimental animal models for evaluating dosage. However, the increasedcell binding affinity and specificity associated with the compounds ofthe present invention permits a wider margin of safety for dosageconcentrations and for repeated dosing.

The pharmaceutical compositions of the present invention can be preparedfor administration by a variety of different routes. In general, thetype of carrier is selected based on the mode of administration.Pharmaceutical compositions can be formulated for any appropriate mannerof administration, including, for example, topical, oral, nasal,intrathecal, rectal, vaginal, sublingual or parenteral administration,including subcutaneous, intravenous, intramuscular, intrastemal,intracavemous, intrameatal, or intraurethral injection or infusion. Apharmaceutical composition (e.g., for oral administration or delivery byinjection) can be in the form of a liquid (e.g., an elixir, syrup,solution, emulsion or suspension). A liquid pharmaceutical compositionmay include, for example, one or more of the following: sterile diluentssuch as water for injection, saline solution, preferably physiologicalsaline, Ringer's solution, isotonic sodium chloride, fixed oils that mayserve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents;antioxidants; chelating agents; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. A parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic. The use of physiological saline is preferred, and an injectablepharmaceutical composition is preferably sterile.

The formulations of the invention are also suitable for administrationin all body spaces/cavities, including but not limited to pleura,peritoneum, cranium, mediastinum, pericardium, bursae or bursal,epidural, intrathecal, intraocular, intra-articular, intra-discal,intra-medullary, perispinal, etc.

Some slow release embodiments include polymeric substances that arebiodegradable and/or dissolve slowly. Such polymeric substances includepolyvinylpyrrolidone, low- and medium-molecular-weight hydroxypropylcellulose and hydroxypropyl methylcellulose, cross-linked sodiumcarboxymethylcellulose, carboxymethyl starch, potassiummethacrylatedivinylbenzene copolymer, polyvinyl alcohols, starches,starch derivatives, microcrystalline cellulose, ethylcellulose,methylcellulose, and cellulose derivatives, (3-cyclodextrin, poly(methylvinyl ethers/maleic anhydride), glucans, scierozlucans, mannans,xanthans, alzinic acid and derivatives thereof, dextrin derivatives,glyceryl monostearate, semisynthetic glycerides, glycerylpalmitostearate, glyceryl behenate, polyvinylpyrrolidone, gelatine,agnesium stearate, stearic acid, sodium stearate, talc, sodium benzoate,boric acid, and colloidal silica.

Slow release agents of the invention may also include adjuvants such asstarch, pregelled starch, calcium phosphate mannitol, lactose,saccharose, glucose, sorbitol, microcrystalline cellulose, gelatin,polyvinylpyrrolidone, methylcellulose, starch solution, ethylcellulose,arabic gum, tragacanth gum, magnesium stearate, stearic acid, colloidalsilica, glyceryl monostearate, hydrogenated castor oil, waxes, andmono-, bi-, and trisubstituted glycerides. Slow release agents may alsobe prepared as generally described in WO94/06416.

In practicing the methods of the present invention, the pharmaceuticalcompositions can be used alone, or in combination with other therapeuticor diagnostic agents. The additional drugs used in the combinationprotocols of the present invention can be administered separately or oneor more of the drugs used in the combination protocols can beadministered together, such as in an admixture. Where one or more drugsare administered separately, the timing and schedule of administrationof each drug can vary. The other therapeutic or diagnostic agents can beadministered at the same time as the compounds of the present invention,separately or at different times.

VI. Methods

In view of the above, the present invention provides, in one aspect, amethod for inhibiting a soluble epoxide hydrolase, comprising contactingthe soluble epoxide hydrolase with an inhibiting amount of a compound ofthe present invention.

In other embodiments, the present invention provides a method formonitoring the activity of a soluble epoxide hydrolase, the methodincluding contacting the soluble epoxide hydrolase with an amount of acompound of the present invention sufficient to produce a detectablechange in the fluorescence of the soluble epoxide hydrolase byinteracting with one or more tryptophan residues present in thecatalytic site of said sEH.

A. Assays to Monitor Soluble Epoxide Hydrolase Activity

Additionally, the present invention provides a variety of assays andassociated methods for monitoring soluble epoxide hydrolase activity,particularly the activity that has been modulated by the administrationof one or more of the compounds provided above.

In one group of embodiments, the invention provides methods for reducingthe formation of a biologically active diol produced by the action of asoluble epoxide hydrolase, the method comprising contacting the solubleepoxide hydrolase with an amount of a compound of the present invention,sufficient to inhibit the activity of the soluble epoxide hydrolase andreduce the formation of the biologically active diol.

In another group of embodiments, the invention provides methods forstabilizing biologically active epoxides in the presence of a solubleepoxide hydrolase, the method comprising contacting the soluble epoxidehydrolase with an amount of a compound of the present invention,sufficient to inhibit the activity of the soluble epoxide hydrolase andstabilize the biologically active epoxide.

In each of these groups of embodiments, the methods can be carried outas part of an in vitro assay or the methods can be carried out in vivoby monitoring blood titers of the respective biologically active epoxideor diol.

Epoxides and diols of some fatty acids are biologically importantchemical mediators and are involved in several biological processes. Thestrongest biological data support the action of oxylipins as chemicalmediators between the vascular endothelium and vascular smooth muscle.Epoxy lipids are anti-inflammatory and anti-hypertensive. Additionally,the lipids are thought to be metabolized by beta-oxidation, as well asby epoxide hydration. The soluble epoxide hydrolase is considered to bethe major enzyme involved in the hydrolytic metabolism of theseoxylipins. The compounds of the present invention can inhibit theepoxide hydrolase and stabilize the epoxy lipids both in vitro and invivo. This activity results in a reduction of hypertension in fourseparate rodent models. Moreover, the inhibitors show a reduction inrenal inflammation associated with and independent of the hypertensivemodels.

More particularly, the present invention provides methods for monitoringa variety of lipids in both the arachidonate and linoleate cascadesimultaneously in order to address the biology of the system. A GLC-MSsystem or a LC-MS method can be used to monitor over 740 analytes in ahighly quantitative fashion in a single injection. The analytes includethe regioisomers of the arachidonate epoxides (EETs), the diols (DHETs),as well as other P450 products including HETEs. Characteristic productsof the cyclooxygenase, lipoxygenase, and peroxidase pathways in both thearachidonate and linoleate series can also be monitored. Such methodsare particularly useful as being predictive of certain disease states.The oxylipins can be monitored in mammals following the administrationof inhibitors of epoxide hydrolase. Generally, EH inhibitors increaseepoxy lipid concentrations at the expense of diol concentrations in bodyfluids and tissues.

Other compounds for use in this aspect of the invention are thosecompounds of the present invention in which the primary pharmacophore isseparated from a secondary and/or tertiary pharmacophore by a distancethat approximates the distance between the terminal carboxylic acid andan epoxide functional group in the natural substrate.

B. Methods of Treating Diseases Modulated by Soluble Epoxide Hydrolases

In another aspect, the present invention provides methods of treatingdiseases, especially those modulated by soluble epoxide hydrolases(sEH). The methods generally involve administering to a subject in needof such treatment an effective amount of a compound of the presentinvention. The dose, frequency and timing of such administering willdepend in large part on the selected therapeutic agent, the nature ofthe condition being treated, the condition of the subject including age,weight and presence of other conditions or disorders, the formulationbeing administered and the discretion of the attending physician.Preferably, the compositions and compounds of the invention and thepharmaceutically acceptable salts thereof are administered via oral,parenteral, subcutaneous, intramuscular, intravenous or topical routes.Generally, the compounds are administered in dosages ranging from about2 mg up to about 2,000 mg per day, although variations will necessarilyoccur depending, as noted above, on the disease target, the patient, andthe route of administration. Dosages are administered orally in therange of about 0.05 mg/kg to about 20 mg/kg, more preferably in therange of about 0.05 mg/kg to about 2 mg/kg, most preferably in the rangeof about 0.05 mg/kg to about 0.2 mg per kg of body weight per day. Thedosage employed for the topical administration will, of course, dependon the size of the area being treated.

It has previously been shown that inhibitors of soluble epoxidehydrolase (“sEH”) can reduce hypertension. See, e.g., U.S. Pat. No.6,351,506. Such inhibitors can be useful in controlling the bloodpressure of persons with undesirably high blood pressure, includingthose who suffer from diabetes.

In some embodiments, compounds of the present invention are administeredto a subject in need of treatment for hypertension, specifically renal,hepatic, or pulmonary hypertension; inflammation, specifically renalinflammation, vascular inflammation, and lung inflammation; adultrespiratory distress syndrome; diabetic complications; end stage renaldisease; Raynaud syndrome and arthritis.

C. Methods for Inhibiting Progression of Kidney Deterioration(Nephropathy) and Reducing Blood Pressure

In another aspect of the invention, the compounds of the invention canreduce damage to the kidney, and especially damage to kidneys fromdiabetes, as measured by albuminuria. The compounds of the invention canreduce kidney deterioration (nephropathy) from diabetes even inindividuals who do not have high blood pressure. The conditions oftherapeutic administration are as described above.

cis-Epoxyeicosantrienoic acids (“EETs”) can be used in conjunction withthe compounds of the invention to further reduce kidney damage. EETs,which are epoxides of arachidonic acid, are known to be effectors ofblood pressure, regulators of inflammation, and modulators of vascularpermeability. Hydrolysis of the epoxides by sEH diminishes thisactivity. Inhibition of sEH raises the level of EETs since the rate atwhich the EETs are hydrolyzed into DHETs is reduced. Without wishing tobe bound by theory, it is believed that raising the level of EETsinterferes with damage to kidney cells by the micro vasculature changesand other pathologic effects of diabetic hyperglycemia. Therefore,raising the EET level in the kidney is believed to protect the kidneyfrom progression from microalbuminuria to end stage renal disease.

EETs are well known in the art. EETs useful in the methods of thepresent invention include 14,15-EET, 8,9-EET and 11,12-EET, and 5,6EETs, in that order of preference. Preferably, the EETs are administeredas the methyl ester, which is more stable. Persons of skill willrecognize that the EETs are regioisomers, such as 8S,9R- and14R,15S-EET. 8,9-EET, 11,12-EET, and 14R,15S-EET, are commerciallyavailable from, for example, Sigma-Aldrich (catalog nos. E5516, E5641,and E5766, respectively, Sigma-Aldrich Corp., St. Louis, Mo.).

EETs produced by the endothelium have anti-hypertensive properties andthe EETs 11,12-EET and 14,15-EET may be endothelium-derivedhyperpolarizing factors (EDHFs). Additionally, EETs such as 11,12-EEThave profibrinolytic effects, anti-inflammatory actions and inhibitsmooth muscle cell proliferation and migration. In the context of thepresent invention, these favorable properties are believed to protectthe vasculature and organs during renal and cardiovascular diseasestates.

It is now believed that sEH activity can be inhibited sufficiently toincrease the levels of EETs and thus augment the effects ofadministering sEH inhibitors by themselves. This permits EETs to be usedin conjunction with one or more sEH inhibitors to reduce nephropathy inthe methods of the invention. It further permits EETs to be used inconjunction with one or more sEH inhibitors to reduce hypertension, orinflammation, or both. Thus, medicaments of EETs can be made which canbe administered in conjunction with one or more sEH inhibitors, or amedicament containing one or more sEH inhibitors can optionally containone or more EETs.

The EETs can be administered concurrently with the sEH inhibitor, orfollowing administration of the sEH inhibitor. It is understood that,like all drugs, inhibitors have half-lives defined by the rate at whichthey are metabolized by or excreted from the body, and that theinhibitor will have a period following administration during which itwill be present in amounts sufficient to be effective. If EETs areadministered after the inhibitor is administered, therefore, it isdesirable that the EETs be administered during the period during whichthe inhibitor will be present in amounts to be effective to delayhydrolysis of the EETs. Typically, the EET or EETs will be administeredwithin 48 hours of administering an sEH inhibitor. Preferably, the EETor EETs are administered within 24 hours of the inhibitor, and even morepreferably within 12 hours. In increasing order of desirability, the EETor EETs are administered within 10, 8, 6, 4, 2, hours, 1 hour, or onehalf hour after administration of the inhibitor. Most preferably, theEET or EETs are administered concurrently with the inhibitor.

In some embodiments, the EETs, the compound of the invention, or both,are provided in a material that permits them to be released over time toprovide a longer duration of action. Slow release coatings are wellknown in the pharmaceutical art; the choice of the particular slowrelease coating is not critical to the practice of the presentinvention.

EETs are subject to degradation under acidic conditions. Thus, if theEETs are to be administered orally, it is desirable that they areprotected from degradation in the stomach. Conveniently, EETs for oraladministration may be coated to permit them to passage the acidicenvironment of the stomach into the basic environment of the intestines.Such coatings are well known in the art. For example, aspirin coatedwith so-called “enteric coatings” is widely available commercially. Suchenteric coatings may be used to protect EETs during passage through thestomach. An exemplary coating is set forth in the Examples.

While the anti-hypertensive effects of EETs have been recognized, EETshave not been administered to treat hypertension because it was thoughtendogenous sEH would hydrolyse the EETs too quickly for them to have anyuseful effect. Surprisingly, it was found during the course of thestudies underlying the present invention that exogenously administeredinhibitors of sEH succeeded in inhibiting sEH sufficiently that levelsof EETs could be further raised by the administration of exogenous EETs.These findings underlie the co-administration of sEH inhibitors and ofEETs described above with respect to inhibiting the development andprogression of nephropathy. This is an important improvement inaugmenting treatment. While levels of endogenous EETs are expected torise with the inhibition of sEH activity caused by the action of the sEHinhibitor, and therefore to result in at least some improvement insymptoms or pathology, it may not be sufficient in all cases to inhibitprogression of kidney damage fully or to the extent intended. This isparticularly true where the diseases or other factors have reduced theendogenous concentrations of EETs below those normally present inhealthy individuals. Administration of exogenous EETs in conjunctionwith a sEH inhibitor is therefore expected to be beneficial and toaugment the effects of the sEH inhibitor in reducing the progression ofdiabetic nephropathy.

The present invention can be used with regard to any and all forms ofdiabetes to the extent that they are associated with progressive damageto the kidney or kidney function. The chronic hyperglycemia of diabetesis associated with long-term damage, dysfunction, and failure of variousorgans, especially the eyes, kidneys, nerves, heart, and blood vessels.The long-term complications of diabetes include retinopathy withpotential loss of vision; nephropathy leading to renal failure;peripheral neuropathy with risk of foot ulcers, amputation, and Charcotjoints.

In addition, persons with metabolic syndrome are at high risk ofprogression to type 2 diabetes, and therefore at higher risk thanaverage for diabetic nephropathy. It is therefore desirable to monitorsuch individuals for microalbuminuria, and to administer a sEH inhibitorand, optionally, one or more EETs, as an intervention to reduce thedevelopment of nephropathy. The practitioner may wait untilmicroalbuminuria is seen before beginning the intervention. As notedabove, a person can be diagnosed with metabolic syndrome without havinga blood pressure of 130/85 or higher. Both persons with blood pressureof 130/85 or higher and persons with blood pressure below 130/85 canbenefit from the administration of sEH inhibitors and, optionally, ofone or more EETs, to slow the progression of damage to their kidneys. Insome embodiments, the person has metabolic syndrome and blood pressurebelow 130/85.

Dyslipidemia or disorders of lipid metabolism is another risk factor forheart disease. Such disorders include an increased level of LDLcholesterol, a reduced level of HDL cholesterol, and an increased levelof triglycerides. An increased level of serum cholesterol, andespecially of LDL cholesterol, is associated with an increased risk ofheart disease. The kidneys are also damaged by such high levels. It isbelieved that high levels of triglycerides are associated with kidneydamage. In particular, levels of cholesterol over 200 mg/dL, andespecially levels over 225 mg/dL, would suggest that sEH inhibitors and,optionally, EETs, should be administered. Similarly, triglyceride levelsof more than 215 mg/dL, and especially of 250 mg/dL or higher, wouldindicate that administration of sEH inhibitors and, optionally, of EETs,would be desirable. The administration of compounds of the presentinvention with or without the EETs, can reduce the need to administerstatin drugs (HMG-CoA reductase inhibitors) to the patients, or reducethe amount of the statins needed. In some embodiments, candidates forthe methods, uses and compositions of the invention have triglyceridelevels over 215 mg/dL and blood pressure below 130/85. In someembodiments, the candidates have triglyceride levels over 250 mg/dL andblood pressure below 130/85. In some embodiments, candidates for themethods, uses and compositions of the invention have cholesterol levelsover 200 mg/dL and blood pressure below 130/85. In some embodiments, thecandidates have cholesterol levels over 225 mg/dL and blood pressurebelow 130/85.

D. Methods of Inhibiting the Proliferation of Vascular Smooth MuscleCells

In other embodiments, compounds of the present invention inhibitproliferation of vascular smooth muscle (VSM) cells without significantcell toxicity (e.g., specific to VSM cells). Because VSM cellproliferation is an integral process in the pathophysiology ofatherosclerosis, these compounds are suitable for slowing or inhibitingatherosclerosis. These compounds are useful to subjects at risk foratherosclerosis, such as individuals who have had a heart attack or atest result showing decreased blood circulation to the heart. Theconditions of therapeutic administration are as described above.

The methods of the invention are particularly useful for patients whohave had percutaneous intervention, such as angioplasty to reopen anarrowed artery, to reduce or to slow the narrowing of the reopenedpassage by restenosis. In some embodiments, the artery is a coronaryartery. The compounds of the invention can be placed on stents inpolymeric coatings to provide a controlled localized release to reducerestenosis. Polymer compositions for implantable medical devices, suchas stents, and methods for embedding agents in the polymer forcontrolled release, are known in the art and taught, for example, inU.S. Pat. Nos. 6,335,029; 6,322,847; 6,299,604; 6,290,722; 6,287,285;and 5,637,113. In some embodiments, the coating releases the inhibitorover a period of time, preferably over a period of days, weeks, ormonths. The particular polymer or other coating chosen is not a criticalpart of the present invention.

The methods of the invention are useful for slowing or inhibiting thestenosis or restenosis of natural and synthetic vascular grafts. Asnoted above in connection with stents, desirably, the synthetic vasculargraft comprises a material which releases a compound of the inventionover time to slow or inhibit VSM proliferation and the consequentstenosis of the graft. Hemodialysis grafts are a particular embodiment.

In addition to these uses, the methods of the invention can be used toslow or to inhibit stenosis or restenosis of blood vessels of personswho have had a heart attack, or whose test results indicate that theyare at risk of a heart attack.

In one group of embodiments, compounds of the invention are administeredto reduce proliferation of VSM cells in persons who do not havehypertension. In another group of embodiments, compounds of theinvention are used to reduce proliferation of VSM cells in persons whoare being treated for hypertension, but with an agent that is not an sEHinhibitor.

The compounds of the invention can be used to interfere with theproliferation of cells which exhibit inappropriate cell cycleregulation. In one important set of embodiments, the cells are cells ofa cancer. The proliferation of such cells can be slowed or inhibited bycontacting the cells with a compound of the invention. The determinationof whether a particular compound of the invention can slow or inhibitthe proliferation of cells of any particular type of cancer can bedetermined using assays routine in the art.

In addition to the use of the compounds of the invention, the levels ofEETs can be raised by adding EETs. VSM cells contacted with both an EETand a compound of the invention exhibited slower proliferation thancells exposed to either the EET alone or to the a compound of theinvention alone. Accordingly, if desired, the slowing or inhibition ofVSM cells of a compound of the invention can be enhanced by adding anEET along with a compound of the invention. In the case of stents orvascular grafts, for example, this can conveniently be accomplished byembedding the EET in a coating along with a compound of the invention sothat both are released once the stent or graft is in position.

E. Methods of Inhibiting the Progression of Obstructive PulmonaryDisease, Interstitial Lung Disease, or Asthma

Chronic obstructive pulmonary disease, or COPD, encompasses twoconditions, emphysema and chronic bronchitis, which relate to damagecaused to the lung by air pollution, chronic exposure to chemicals, andtobacco smoke. Emphysema as a disease relates to damage to the alveoliof the lung, which results in loss of the separation between alveoli anda consequent reduction in the overall surface area available for gasexchange. Chronic bronchitis relates to irritation of the bronchioles,resulting in excess production of mucin, and the consequent blocking bymucin of the airways leading to the alveoli. While persons withemphysema do not necessarily have chronic bronchitis or vice versa, itis common for persons with one of the conditions to also have the other,as well as other lung disorders.

Some of the damage to the lungs due to COPD, emphysema, chronicbronchitis, and other obstructive lung disorders can be inhibited orreversed by administering inhibitors of the enzyme known as solubleepoxide hydrolase, or “sEH”. The effects of sEH inhibitors can beincreased by also administering EETs. The effect is at least additiveover administering the two agents separately, and may indeed besynergistic.

The studies reported herein show that EETs can be used in conjunctionwith sEH. inhibitors to reduce damage to the lungs by tobacco smoke or,by extension, by occupational or environmental irritants. These findingsindicate that the co-administration of sEH inhibitors and of EETs can beused to inhibit or slow the development or progression of COPD,emphysema, chronic bronchitis, or other chronic obstructive lungdiseases which cause irritation to the lungs.

Animal models of COPD and humans with COPD have elevated levels ofimmunomodulatory lymphocytes and neutrophils. Neutrophils release agentsthat cause tissue damage and, if not regulated, will over time have adestructive effect. Without wishing to be bound by theory, it isbelieved that reducing levels of neutrophils reduces tissue damagecontributing to obstructive lung diseases such as COPD, emphysema, andchronic bronchitis. Administration of sEH inhibitors to rats in ananimal model of COPD resulted in a reduction in the number ofneutrophils found in the lungs. Administration of EETs in addition tothe sEH inhibitors also reduced neutrophil levels. The reduction inneutrophil levels in the presence of sEH inhibitor and EETs was greaterthan in the presence of the sEH inhibitor alone.

While levels of endogenous EETs are expected to rise with the inhibitionof sEH activity caused by the action of the sEH inhibitor, and thereforeto result in at least some improvement in symptoms or pathology, it maynot be sufficient in all cases to inhibit progression of COPD or otherpulmonary diseases. This is particularly true where the diseases orother factors have reduced the endogenous concentrations of EETs belowthose normally present in healthy individuals. Administration ofexogenous EETs in conjunction with an sEH inhibitor is thereforeexpected to augment the effects of the sEH inhibitor in inhibiting orreducing the progression of COPD or other pulmonary diseases.

In addition to inhibiting or reducing the progression of chronicobstructive airway conditions, the invention also provides new ways ofreducing the severity or progression of chronic restrictive airwaydiseases. While obstructive airway diseases tend to result from thedestruction of the lung parenchyma, and especially of the alveoli,restrictive diseases tend to arise from the deposition of excesscollagen in the parenchyma. These restrictive diseases are commonlyreferred to as “interstitial lung diseases”, or “ILDs”, and includeconditions such as idiopathic pulmonary fibrosis. The methods,compositions and uses of the invention are useful for reducing theseverity or progression of ILDs, such as idiopathic pulmonary fibrosis.Macrophages play a significant role in stimulating interstitial cells,particularly fibroblasts, to lay down collagen. Without wishing to bebound by theory, it is believed that neutrophils are involved inactivating macrophages, and that the reduction of neutrophil levelsfound in the studies reported herein demonstrate that the methods anduses of the invention will also be applicable to reducing the severityand progression of ILDs.

In some embodiments, the ILD is idiopathic pulmonary fibrosis. In otherembodiments, the ILD is one associated with an occupational orenvironmental exposure. Exemplars of such ILDs, are asbestosis,silicosis, coal worker's pneumoconiosis, and berylliosis. Further,occupational exposure to any of a number of inorganic dusts and organicdusts is believed to be associated with mucus hypersecretion andrespiratory disease, including cement dust, coke oven emissions, mica,rock dusts, cotton dust, and grain dust (for a more complete list ofoccupational dusts associated with these conditions, see Table 254-1 ofSpeizer, “Environmental Lung Diseases,” Harrison's Principles ofInternal Medicine, infra, at pp. 1429-1436). In other embodiments, theILD is sarcoidosis of the lungs. ILDs can also result from radiation inmedical treatment, particularly for breast cancer, and from connectivetissue or collagen diseases such as rheumatoid arthritis and systemicsclerosis. It is believed that the methods, uses and compositions of theinvention can be useful in each of these interstitial lung diseases.

In another set of embodiments, the invention is used to reduce theseverity or progression of asthma. Asthma typically results in mucinhypersecretion, resulting in partial airway obstruction. Additionally,irritation of the airway results in the release of mediators whichresult in airway obstruction. While the lymphocytes and otherimmunomodulatory cells recruited to the lungs in asthma may differ fromthose recruited as a result of COPD or an ILD, it is expected that theinvention will reduce the influx of immunomodulatory cells, such asneutrophils and eosinophils, and ameliorate the extent of obstruction.Thus, it is expected that the administration of sEH inhibitors, and theadministration of sEH inhibitors in combination with EETs, will beuseful in reducing airway obstruction due to asthma.

In each of these diseases and conditions, it is believed that at leastsome of the damage to the lungs is due to agents released by neutrophilswhich infiltrate into the lungs. The presence of neutrophils in theairways is thus indicative of continuing damage from the disease orcondition, while a reduction in the number of neutrophils is indicativeof reduced damage or disease progression. Thus, a reduction in thenumber of neutrophils in the airways in the presence of an agent is amarker that the agent is reducing damage due to the disease orcondition, and is slowing the further development of the disease orcondition. The number of neutrophils present in the lungs can bedetermined by, for example, bronchoalveolar lavage.

F. Prophylactic and Therapeutic Methods to Reduce Stroke Damage

Inhibitors of soluble epoxide hydrolase (“sEH”) and EETs administered inconjunction with inhibitors of sEH have been shown to reduce braindamage from strokes. Based on these results, we expect that inhibitorsof sEH taken prior to an ischemic stroke will reduce the area of braindamage and will likely reduce the consequent degree of impairment. Thereduced area of damage should also be associated with a faster recoveryfrom the effects of the stroke.

While the pathophysiologies of different subtypes of stroke differ, theyall cause brain damage. Hemorrhagic stroke differs from ischemic strokein that the damage is largely due to compression of tissue as bloodbuilds up in the confined space within the skull after a blood vesselruptures, whereas in ischemic stroke, the damage is largely due to lossof oxygen supply to tissues downstream of the blockage of a blood vesselby a clot. Ischemic strokes are divided into thrombotic strokes, inwhich a clot blocks a blood vessel in the brain, and embolic strokes, inwhich a clot formed elsewhere in the body is carried through the bloodstream and blocks a vessel there. But, in both hemorrhagic stroke andischemic stroke, the damage is due to the death of brain cells. Based onthe results observed in our studies, however, we would expect at leastsome reduction in brain damage in all types of stroke and in allsubtypes.

A number of factors are associated with an increased risk of stroke.Given the results of the studies underlying the present invention, sEHinhibitors administered to persons with any one or more of the followingconditions or risk factors: high blood pressure, tobacco use, diabetes,carotid artery disease, peripheral artery disease, atrial fibrillation,transient ischemic attacks (TIAs), blood disorders such as high redblood cell counts and sickle cell disease, high blood cholesterol,obesity, alcohol use of more than one drink a day for women or twodrinks a day for men, use of cocaine, a family history of stroke, aprevious stroke or heart attack, or being elderly, will reduce the areaof brain damaged of a stroke. With respect to being elderly, the risk ofstroke increases for every 10 years. Thus, as an individual reaches 60,70, or 80, administration of sEH inhibitors has an increasingly largerpotential benefit. As noted in the next section, the administration ofEETs in combination with one or more sEH inhibitors can be beneficial infurther reducing the brain damage. One can expect beneficial effectsfrom sEHI with or without EETs in a variety of diseases which lead toischemia reperfusion injury such as heart attacks.

In some uses and methods, the sEH inhibitors and, optionally, EETs, areadministered to persons who use tobacco, have carotid artery disease,have peripheral artery disease, have atrial fibrillation, have had oneor more transient ischemic attacks (TIAs), have a blood disorder such asa high red blood cell count or sickle cell disease, have high bloodcholesterol, are obese, use alcohol in excess of one drink a day if awoman or two drinks a day if a man, use cocaine, have a family historyof stroke, have had a previous stroke or heart attack and do not havehigh blood pressure or diabetes, or are 60, 70, or 80 years of age ormore and do not have hypertension or diabetes.

Clot dissolving agents, such as tissue plasminogen activator (tPA), havebeen shown to reduce the extent of damage from ischemic strokes ifadministered in the hours shortly after a stroke. tPA, for example, isapproved by the FDA for use in the first three hours after a stroke.Thus, at least some of the brain damage from a stroke is notinstantaneous, but occurs over a period of time or after a period oftime has elapsed after the stroke. It is therefore believed thatadministration of sEH inhibitors, optionally with EETs, can also reducebrain damage if administered within 6 hours after a stroke has occurred,more preferably within 5, 4, 3, or 2 hours after a stroke has occurred,with each successive shorter interval being more preferable. Even morepreferably, the inhibitor or inhibitors are administered 2 hours or lessor even 1 hour or less after the stroke, to maximize the reduction inbrain damage. Persons of skill are well aware of how to make a diagnosisof whether or not a patient has had a stroke. Such determinations aretypically made in hospital emergency rooms, following standarddifferential diagnosis protocols and imaging procedures.

In some uses and methods, the sEH inhibitors and, optionally, EETs, areadministered to persons who have had a stroke within the last 6 hourswho: use tobacco, have carotid artery disease, have peripheral arterydisease, have atrial fibrillation, have had one or more transientischemic attacks (TIAs), have a blood disorder such as a high red bloodcell count or sickle cell disease, have high blood cholesterol, areobese, use alcohol in excess of one drink a day if a woman or two drinksa day if a man, use cocaine, have a family history of stroke, have had aprevious stroke or heart attack and do not have high blood pressure ordiabetes, or are 60, 70, or 80 years of age or more and do not havehypertension or diabetes.

The conditions of therapeutic administration for all of theseindications are as described above.

The following examples are provided to illustrate the invention and arenot intended to limit any aspect of the invention as set forth above orin the claims below.

VII. Examples Example 1 Synthesis of Compounds 1-59

General.

All reagents and solvents were purchased from commercial suppliers andwere used without further purification. All reactions were performedunder an inert atmosphere of dry nitrogen. Flash chromatography wasperformed on silica gel using a dry loading technique, where necessaryfor poorly soluble products, and elution with the appropriate solventsystem. Melting points were determined using an OptiMelt melting pointapparatus and are uncorrected. ¹H-NMIR spectra were collected using aBruker Avance 500 MHz spectrometer or Varian Mercury 300 MHzspectrometer. Signal multiplicities are represented as singlet (s),doublet (d), double doublet (dd), triplet (t), quartet (q), quintet(quint), multiplet (m), broad (br), broad singlet (brs), broad doublet(br d), broad triplet (br t), broad multiplet (br m), doublet of doubletof doublets (ddd) and quartet of doublets (qd). Accurate masses weremeasured using a Micromass LCT ESI-TOF-MS equipped with a Waters 2795HPLC. Log P and purity analyses were performed using a Hewlett Packard1100 HPLC equipped with a diode array detector. A Phenomenex Luna 150mm×4.6 mm, 5 μm, C-18 column was used for all HPLC analyses.

The abbreviations used in the examples below have the following meaning:melting point (Mp), mass spectroscopy (MS), thin layer chromatography(TLC), the parent peak in the MS plus H⁺ ([M+H]⁺), minute (min),kilogram (kg), milligram (mg), nanomolar (nM), tetrahydrofuran (THF),tertiary butoxy carbonyl (BOC), potassium sulfate (KHSO₄), potassiumhydroxide (KOH), magnesium sulfate (MgSO⁴), hydrogen chloride (HCl),dimethylsulfoxide (DMSO), ethyl (Et), ethyl acetate (EtOAc), methanol(MeOH), dichloromethane (CH₂Cl₂, DCM), area under the concentration(AUC).

Log P Determination.

Octanol-water partition coefficients were determined by an HPLC methodfollowing OECD guideline 117. The accepted error for this method is ±0.5of shake flask values. Isocratic MeOH:H₂O (3:1, v/v), 50 mM ammoniumacetate in MeOH:H₂O (3:1, v/v) adjusted to pH 9.0, and MeOH:H₂O (3:1,v/v) adjusted to pH 3.0 with H₃PO₄ were used for neutral, basic andacidic analytes, respectively, with a flow rate of 0.75 mL/min. The HPLCmethod was validated using compounds 24 and 54, which were found to havelog P values of 1.9 and 2.3, respectively, using the shake flask method(OECD guideline 107).

Purity Determination.

Final products were dissolved in MeOH:H₂O (3:1, v/v) at 10 μg/mL, and100 μL injections were analyzed in triplicate by HPLC-UV with detectionat 210 nm, 230 nm, 254 nm and 290 nm. HPLC conditions were the same asthose for log P determination. Purity was judged as the percent of totalpeak area for each wavelength. The lowest observed purity is reported.Compounds were also judged to be pure based on thin layer chromatographyvisualized with short wave UV and stained with basic potassiumpermanganate.

Method A—Synthesis of Aryl and Alkyl Isocyanates.

The aniline or amine (1 mmol) was added to an ice cold, stirred biphasicmixture of DCM (10 mL) and saturated sodium bicarbonate (10 mL), or 1NNaOH (3 mL) in brine (7 mL) where noted. Stirring was stoppedmomentarily, triphosgene (0.37 eq.) in DCM (1 mL) added via syringe tothe lower DCM layer and stirring continued for 10 minutes. The DCM layerwas removed and filtered through a bed of magnesium sulfate. Thefiltrate was evaporated to afford the corresponding isocyanate, whichwas used without further purification.

Method B—Synthesis of Ureas Via Isocyanate.

The isocyanate (1 mmol) was dissolved or suspended in dry THF (3-5 mL)and cooled in an ice bath. The amine (1 mmol) was dissolved in dry THF(1 mL) and slowly added to the reaction. Stirring was continued for 1 to24 hours at rt. The reaction was quenched with dilute HCl (or waterwhere the BOC group was present) and extracted into ethyl acetate. Thecombined organic phase was dried, evaporated and purified.

Method C—Synthesis of Ureas Via 4-Nitrophenylcarbamate.

To an ice cold solution of 4-nitrophenyl chloroformate (1 eq) in dry THFwas added Et₃N (1.3 eq) and the appropriate aniline (1 eq) dissolved indry THF. The reaction was allowed to warm to rt, stirred for 30 minutesand then filtered. The filtrate was evaporated and dissolved in DMF.Amine 1 was added and the reaction warmed to 50° C. for 1-3 hours. Themixture was cooled to rt, diluted with ethyl acetate, and the organicphase washed with 1N NaOH until the wash was free of yellowp-nitrophenol. The organic phase was dried, evaporated and purified.

Method D—Synthesis of N-Acyl Piperidine Analogues.

To a solution of 41 (1 eq) in DCM was added the corresponding carboxylicacid (1.1 eq), DMAP (1 eq) and EDCI (1.1 eq). The reaction was stirredfor 12-24 hours at rt, and neutral products worked up by partition withEtOAc and IN HCl (basic products by partition with saturated sodiumbicarbonate and EtOAc) and the organic phase was dried, evaporated andpurified.

Method E—Synthesis of N-Sulfonyl Piperidine Analogues.

To a solution of 41 (152 mg, 0.5 mmol) in dry THF (5 mL) was added Et₃N(1.3 eq) and the corresponding sulfonyl chloride (1 eq) in dry THF (1mL). The reaction was stirred for 12 hours, quenched with IN HCl andfiltered to collect the resulting precipitate, which was furtherpurified.

1-(4-Aminopiperidin-1-yl)propan-1-one. (1)

4-Aminopiperidine (4.01 g, 40 mmol) and benzaldehyde (4.251 g, 40 mmol)were dissolved in toluene (70 mL) and refluxed on a Dean-Stark trapuntil water ceased to evolve. The solvent was evaporated and the residuewas reconstituted in dry THF (75 mL). Triethylamine (4.04 g, 40 mmol)was added and the reaction was cooled in an ice bath. With vigorousstirring, propionyl chloride (3.70 g, 40 mmol) was added and thereaction continued for 1.5 hours at rt. The reaction was filtered, andthe filtrate was evaporated and treated with IN HCl (50 mL) for 1 hour.The aqueous phase was washed with Et₂O (3×50 mL), basified to pH>10 withNaOH, saturated with sodium chloride and extracted with DCM (5×75 mL).The combined DCM extract was evaporated and azeotropically dried withtoluene to give intermediate 1 (4.74 g, 64%) as a light brown oil: ¹HNMR (500 MHz, DMSO-d₆) δ 4.18 (d, J=11.8 Hz, 1H), 3.74 (d, J=11.8 Hz,1H), 3.00 (dd, J=11.8, 11.8 Hz, 1H), 2.89 (brs, 2H), 2.82-2.15 (m, 1H),2.67 (dd, J=11.8 Hz, 11.8 Hz, 1H), 2.28 (q, J=7.1 Hz, 2H), 1.72 (d,J=11.8 Hz, 1H), 1.67 (d, J=11.8 Hz, 1H), 1.14 (q, J=10.2 Hz, 1H), 1.04(q, J=10.2 Hz, 1H), 0.98 (t, J=7.1 Hz, 3H).

1-(1-Adamantyl)-3-(1-propionylpiperidin-4-yl)urea. (2)

Prepared according to the procedure in Bioorg. Med. Chem. Lett., 2006,16, 5212-5216. Mp 217-221° C. ¹HNMR (500 MHz, DMSO-d₆) δ 5.67 (d, J=7.6Hz, 1H), 5.41 (s, 1H), 4.10 (dd, J=12.5 Hz, 1H), 3.69 (d, J=12.5 Hz,1H), 3.55-3.46 (m, 1H), 3.07 (dd, J=11.5, 11.5 Hz, 1H), 2.79-2.72 (m,1H), 2.29 (q, J=7.4 Hz, 2H), 1.98 (s, 3H), 1.84 (s, 6H), 1.76 (d, J=12.5Hz, 1H), 1.70 (d, J=12.5 Hz, 1H), 1.60 (brs, 6H), 1.16 (q, J=11.0 Hz,1H), 11.06 (q, J=11.0 Hz, 1H), 0.97 (t, J=7.4 Hz, 3H). Purity 95%. HRMScalculated for C₁₉H₃₁N₃O₂+H⁺ 334.2494. found (ESI(+), [M+H]) 334.2489.

1-Cycloheptyl-3-(1-propionyIpiperidin-4-yl)urea. (3)

Cycloheptyl isocyanate was reacted with 1 by Method B. Flashchromatography eluted with 15:1 EtOAc:MeOH afforded compound 3 (66 mg,22%) as a white solid: Mp 164-172° C. ¹H NMR (500 MHz, DMSO-d₆) δ 5.69(d, 7.7 Hz, 1H), 5.66 (d, J=7.8 Hz, 1H), 4.13 (d, J=12.8 Hz, 1H), 3.71(d, J=13.4 Hz, 1H), 3.61-3.52 (m, 2H), 3.07 (t, J=11.7 Hz, 1H), 2.74 (t,J=11.6 Hz, 1H), 2.29 (q, J=7.4 Hz, 2H), 1.80-1.68 (m, 4H), 1.58-1.42 (m,6H), 1.42-1.28 (m, 4H), 1.25-1.04 (m, 2H), 0.97 (t, J=7.4 Hz, 3H).Purity 92%. HRMS calculated for C¹⁶H₂₉N₃O₂−H⁺ 294.2182. found (ESI(−),[M−H]) 294.2205.

1-Cyclohexyl-3-(1-propionylpiperidin-4-yl)urea. (4)

Cyclohexyl isocyanate was reacted with 1 by Method B. Flashchromatography eluted with EtOAc:MeOH (15:1, v:v) afforded compound 4(63 mg, 22%) as a white solid: Mp 177-179° C. ¹H NMR (500 MHz, DMSO-d₆)δ 5.70 (d, J=7.6 Hz, 1H), 5.62 (d, J=7.9 Hz, 1H), 4.13 (d, J=12.4 Hz,1H), 3.71 (d, J=14.1 Hz, 1H), 3.57 (s, 1H), 3.34 (s, 1H), 3.07 (t,J=11.8 Hz, 1H), 2.74 (t, J=Hz, 1H), 2.29 (q, J=7.4 Hz, 2H), 1.81-1.67(m, 4H), 1.66-1.57 (m, 2H), 1.54-1.46 (m, 1H), 1.30-1.00 (m, 7H), 0.97(t, J=7.4 Hz, 3H). Purity 97%. HRMS calculated for C₁₅H₂₇N₃O₂+H⁺282.2181. found (ESI(+), [M+H]) 282.2146.

1-Octyl-3-(1-propionylpiperidin-4-yl)urea. (5)

Octyl isocyanate was prepared from octylamine by Method A and wassubsequently reacted with 1 by Method B. Flash chromatography elutedwith EtOAc:MeOH (15:1, v:v) afforded compound 5 (145 mg, 47%) as a whitesolid: Mp 131-132° C. ¹H NMR (500 MHz, DMSO-d₆) δ 5.76 (d, J=7.7 Hz,1H), (t, J=5.2 Hz, 1H), 4.15 (d, J=12.4 Hz, 1H), 3.72 (d, J=13.5 Hz,1H), 3.61-3.52 (m, 1H), 3.07 (t, J=12.0 Hz, 1H), 2.95 (dd, J=6.5 Hz,2H), 2.73 (t, J=11.7 Hz, 1H), 2.29 (dd, J=14.8, 7.4 Hz, 2H), 1.80-1.67(m, 1H), 1.38-1.05 (m, 7H obscured by s, 1.24), 1.24 (s, 8H), 0.97 (t,J=7.4 Hz, 3H), 0.86 (t, J=6.7 Hz, 3H). HRMS calculated for C₁₇H₃₃N₃O₂+H⁺312.2651. found (ESI(+), [M+H]) 312.2601.

1-(trans-2-Phenylcyclopropyl)-3-(1-propionylpiperidin-4-yl)urea. (6)

trans-2-Phenylcyclopropyl isocyanate was reacted with 1 by Method B.Flash chromatography eluted with EtOAc:MeOH (15:1, v:v) andrecrystallization from EtOAc:acetone afforded compound 6 (178 mg, 57%)as a white solid: Mp 155-158° C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.24 (t,J=7.5 Hz, 2H), 7.14 (t, J=7.4 Hz, 1H), 7.08 (d, J=7.3 Hz, 2H), 6.23 (s,1H), 5.77 (d, J=7.0 Hz, 1H), 4.16 (d, J=12.9 Hz, 1H), 3.72 (d, J=13.6Hz, 1H), 3.64-3.54 (m, 1H), 3.06 (t, J=12.0 Hz, 1H), 2.71 (t, J=11.9 Hz,1H), 2.62 (dt, J=7.3, 3.5 Hz, 1H), 2.29 (q, J=7.4 Hz, 2H), 1.87 (ddd,J=9.0, 6.0, 3.2 Hz, 1H), 1.75 (dd, J=26.8, 11.7 Hz, 2H), 1.23 (q, J=11.6Hz, 1H), 1.18-1.01 (m, 3H), 0.97 (t, J=7.4 Hz, 3H). Purity 94%. HRMScalculated for C₁₈H₂₅N₃O₂−H⁺ 314.1869. found (ESI(−), [M−H]) 314.1877.

1-Phenyl-3-(1-propionylpiperidin-4-yl)urea. (7)

Phenyl isocyanate was reacted with 1 by Method B. Flash chromatographyeluted with 15:1 EtOAc:MeOH afforded compound 6 (112 mg, 41%) as a whitesolid: Mp 169-171° C. ¹H NMR (500 MHz, DMSO-dg) δ 8.32 (s, 1H), 7.37 (d,J=8.0 Hz, 2H), 7.21 (t, J=7.8 Hz, 1H), 6.88 (t, J=7.4 Hz, 1H), 6.16 (d,J=7.6 Hz, 1H), 4.18 (d, J=12.7 Hz, 1H), 3.80-3.63 (m, 2H), 3.12 (t,J=12.0 Hz, 1H), 2.79 (t, J=11.6 Hz, 1H), 2.32 (dd, J=7.4 Hz, 2H),1.88-1.75 (m, 3H), 1.35-1.13 (m, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity95%. FIRMS calculated for C₁₅H₂₁N₃O₂+H⁺ 276.1712. found (ESI(+), [M+H])276.1658.

1-(Naphthalen-2-yl)-3-(1-propionyIpiperidin-4-yl)urea. (8)

2-Naphthyl isocyanate was prepared from 2-naphthylamine by Method A andwas subsequently reacted with 1 by Method B. Flash chromatography elutedwith 17:1 EtOAc:MeOH afforded compound 8 (159 mg, 49%) as a white solid:Mp 213-215° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.48 (s, 1H), 7.98 (s, 1H),7.76 (d, J=8.6 Hz, 2H), 7.71 (d, J=8.1 Hz, 1H), 7.46-7.38 (m, 2H), 7.30(t, J=7.3 Hz, 1H), 6.22 (d, J=7.4 Hz, 1H), 4.17 (s, 1H), 3.75 (s, 2H),3.17 (s, 1H), 2.84 (s, 1H), 2.32 (dd, J=14.8, 7.5 Hz, 2H), 1.86 (s, 2H),1.42-1.19 (m, 2H), 1.00 (t, J=7.37 Hz, 3H). Purity 96%>. HRMS calculatedfor C₁₉H₂₃N₃O₂−H⁺ 324.1712. found (ESI(−), [M−H]) 324.1683.

1-(1-Propionylpiperidin-4-yl)-3-(pyridin-3-yl)urea. (9)

Pyridine-3-isocyanate was reacted with 1 by Method B. Flashchromatography eluted with 9:1 EtOAc:MeOH afforded compound 9 (266 mg,96%) as a colorless oil: ¹H NMR (500 MHz, DMSO-d₆) δ 8.52 (d, J=Hz, 1H),8.47 (s, 1H), 8.11 (d, J=4.5 Hz, 1H), 7.86 (d, J=8.8 Hz, 1H), 7.24 (dd,J=4.7 Hz, 1H), 6.29 (d, J=7.5 Hz, 1H), 4.18 (d, J=11.9 Hz, 1H),3.81-3.67 (m, 2H), 3.19-3.09 (m, 1H), 2.80 (t, J=11.6 Hz, 1H), 2.32 (q,J=7.4 Hz, 2H), 1.83 (dd, J=25.1, 12.3 Hz, 2H), 1.39-1.20 (m, 2H), 1.18(t, J=7.1 Hz, 3H). Purity 83% by ¹H-NMR. HRMS calculated forC₁₄H₂₀N₄O₂−H⁺ 275.1508. found (ESI(−), [M−H]) 275.1511.

1-(1-Propionylpiperidin-4-yI)-3-o-tolylurea. (10)

o-Tolyl isocyanate was prepared from o-toluidine by Method A and wassubsequently reacted with 1 by Method B. Flash chromatography elutedwith 16:1 EtOAc:MeOH afforded compound 10 (88 mg, 30%) as a white solid:Mp 178-183° C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.84 (d, J=8.1 Hz, 1H), 7.55(s, 1H), 7.13-7.05 (m, 2H), 6.85 (t, J=7.4 Hz, 1H), 6.60 (d, J=7.3 Hz,1H), 4.16 (d, J=13.1 Hz, 1H), 3.75 (d, J=13.8 Hz, 1H), 3.72-3.65 (m,1H), 3.14 (t, J=11.4 Hz, 1H), 2.82 (t, J=11.1 Hz, 1H), 2.32 (dd, J=14.8,7.4 Hz, 2H), 2.17 (s, 3H), 1.90-1.77 (m, 2H), 1.35-1.14 (m, 1H), 0.99(t, J=7.3 Hz, 3H). Purity 95%. HRMS calculated for C₁₆H₂₃N₃O₂+H⁺290.1868. found (ESI(+), [M+H]) 290.1822.

1-(1-Propionylpiperidin-4-yl)-3-m-tolylurea. (11)

m-Tolyl isocyanate was prepared from m-toluidine by Method A and wassubsequently reacted with 1 by Method B. Flash chromatography elutedwith 15:1 EtOAc:MeOH and recrystallization from EtOAc:MeOH affordedcompound 11 (74 mg, 26%) as a white solid: Mp 173-175° C. ¹H NMR (500MHz, DMSO-d₆) δ 8.23 (s, 1H), 7.20 (s, 1H), 7.15 (d, J=8.3 Hz, 1H), 7.08(t, J=7.7 Hz, 1H), 6.70 (d, J=7.3 Hz, 1H), 6.13 (d, J=7.5 Hz, 1H), 4.17(d, J=13.5 Hz, 1H), 3.75 (d, J=13.9 Hz, 1H), 3.72-3.63 (m, 1H), 3.12 (t,J=11.8 Hz, 1H), 2.79 (t, J=11.5 Hz, 1H), 2.31 (q, J=7.4 Hz, 2H), 2.23(s, 3H), 1.88-1.75 (m, 2H), 1.34-1.13 (m, 1H), 0.98 (t, J=7.4 Hz, 3H).Purity 98%. HRMS calculated for C₁₆H₂₃N₃O₂−H⁺ 288.1712. found (ESI(−),[M−H]) 288.1693.

1-(1-Propionylpiperidin-4-yl)-3-p-tolylurea. (12)

p-Tolyl isocyanate was prepared from p-toluidine by Method A and wassubsequently reacted with 1 by Method B. Flash chromatography elutedwith EtOAc afforded compound 12 (81 mg, 28%) as a white solid: Mp180-182° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.20 (s, 1H), 7.25 (d, J=8.3 Hz,2H), 7.01 (d, J=8.1 Hz, 2H), 6.09 (d, J=7.5 Hz, 1H), 4.17 (d, J=13.5 Hz,1H), 3.75 (d, J=14.1 Hz, 1H), 3.71-3.63 (m, 1H), 3.12 (t, J=11.6 Hz,1H), 2.78 (t, J=11.2 Hz, 1H), 2.31 (q, J=7.4 Hz, 2H), 2.21 (s, 3H),1.88-1.74 (m, 2H), 1.34-1.14 (m, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity97%. HRMS calculated for C₁₆H₂₃N₃O₂−H⁺ 288.1712. found (ESI(−), [M−H])288.1716.

1-(4-Ethylphenyl)-3-(1-propionylpiperidin-4-yl)urea. (13)

4-Ethylphenyl isocyanate was prepared from 4-ethylaniline by Method Aand was subsequently reacted with 1 by Method B. Flash chromatographyeluted with 17:1 EtOAc:MeOH and recrystallization from acetone affordedcompound 13 (46 mg, 15%) as a white solid: Mp 164-165° C. ¹H NMR (500MHz, DMSO-d₆) δ 8.21 (s, 1H), 7.27 (d, J=8.4 Hz, 2H), 7.04 (d, J=8.4 Hz,2H), 6.10 (d, J=7.6 Hz, 1H), 4.17 (d, J=13.6 Hz, 1H), 3.75 (d, J=12.9Hz, 1H), 3.72-3.63 (m, 1H), 3.12 (t, J=12.1 Hz, 1H), 2.79 (t, J=10.9 Hz,1H), 2.32 (q, J=7.4 Hz, 2H), 1.88-1.74 (m, 2H), 1.34-1.16 (m, 2H), 1.13(t, J=7.6 Hz, 3H), 0.98 (t, J=7.4 Hz, 3H). Purity 97%. HRMS calculatedfor C₁₇H₂₅N₃O₂+H⁺ 304.2025. found (ESI(+), [M+H]) 304.2005.

1-(4-Isopropylphenyl)-3-(1-propionylpiperidin-4-yl)urea. (14)

4-Isopropylphenyl isocyanate was prepared from 4-isopropylaniline byMethod A and was subsequently reacted with 1 by Method B. Flashchromatography eluted with 15:1 EtOAc:MeOH and recrystallization fromacetone afforded compound 14 (44 mg, 14%) as a white solid. Mp 173-174°C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.25-8.18 (m, 1H), 7.27 (d, J=8.4 Hz,2H), 7.08 (d, J=8.4 Hz, 2H), 6.10 (d, J=7.6 Hz, 1H), 4.17 (d, J=13.2 Hz,1H), 3.75 (d, J=13.9 Hz, 1H), 3.71-3.63 (m, 1H), 3.16-3.07 (m, 1H),2.83-2.74 (m, 2H), 2.32 (dd, J=7.4 Hz, 2H), 1.87-1.75 (m, 2H), 1.34-1.18(m, 2H), 1.16 (d, J=6.9 Hz, 6H), 0.98 (t, J=7.4, 7.4 Hz, 3H). Purity98%. HRMS calculated for C₁₈H₂₇N₃O₂−H⁺ 316.2025. found (ESI(−), [M−HJ)316.1981.

1-(4-Methoxyphenyl)-3-(1-propionylpiperidin-4-yl)urea. (15)

4-Methoxyphenyl isocyanate was prepared from p-anisidine by Method A andwas subsequently reacted with 1 by Method B. Flash chromatography elutedwith 15:1 EtOAc:MeOH afforded compound 15 (82 mg, 27%) as a white solid:Mp 164-165° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.12 (s, 1H), 7.27 (d, J=8.8Hz, 2H), 6.80 (d, J=8.8 Hz, 2H), 6.04 (d, J=7.6 Hz, 1H), 4.17 (d, J=12.9Hz, 1H), 3.75 (d, J=13.5 Hz, 1H), 3.69 (s, 3H), 3.68-3.63 (m, 1H,obscured by s δ 3.69), 3.11 (t, J=12.1 Hz, 1H), 2.78 (dd, J=11.4, 11.4Hz, 1H), 2.31 (dd, J=14.7, 7.3 Hz, 2H), 1.81 (dd, J=26.1, 11.4 Hz, 2H),1.34-1.14 (m, 2H), 0.98 (t, J=7.3 Hz, 3H). Purity 95%. HRMS calculatedfor C₁₆H₂₃N₃O₃+H⁺ 306.1817. found (ESI(+), [M+H]) 306.1780.

1-(4-Phenoxyphenyl)-3-(1-propionylpiperidin-4-yl)urea. (16)

4-Phenoxyphenyl isocyanate was prepared from 4-phenoxyaniline by MethodA and was subsequently reacted with 1 by Method B. Flash chromatographyeluted with EtOAc afforded compound 16 (191 mg, 52%) as a white solid:Mp 153-154° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.35 (s, 1H), 7.39 (d, J=7.9Hz, 2H), 7.34 (t, J=7.9, 7.9 Hz, 2H), 7.07 (t, J=73 Hz, 1H), 6.92 (d,J=6.8 Hz, 4H), 6.14 (d, J=7.5 Hz, 1H), 4.18 (d, J=12.1 Hz, 1H), 3.76 (d,J=12.8 Hz, 1H), 3.72-3.64 (m, 1H), 3.12 (t, J=12.3 Hz, 1H), 2.79 (t,J=11.7 Hz, 1H), 2.32 (q, 0.7=7.4 Hz, 2H), 1.88-1.76 (m, 2H), 1.35-1.15(m, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity 96%. HRMS calculated forC21H25N₃O₃+H⁺ 368.1974. found (ESI(+), [M+H]) 368.1937.

1-(3,4-Methylenedioxyphenyl)-3-(1-propionylpiperidin-4-yl)urea. (17)

3,4-Methylenedioxyaniline (274 mg, 2 mmol) was subject to Method C togive the desired urea via an intermediate 4-nitrophenyl carbamate. Flashchromatography eluted with EtOAc afforded compound 17 (238 mg, 37%) as awhite solid: Mp 195-197° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.16 (s, 1H),7.14 (d, J=1.5 Hz, 1H), 6.75 (d, J=8.3 Hz, 1H), 6.65 (dd, J=8.4, 1.6 Hz,1H), 6.03 (d, J=7.6 Hz, 1H), 5.92 (s, 1H), 4.16 (d, J=12.3 Hz, 1H), 3.74(d, J=12.5 Hz, 1H), 3.71-3.62 (m, 1H), 3.12 (t, J=11.6 Hz, 1H), 2.79 (t,J=11.7 Hz, 1H), 2.31 (q, J=7.4 Hz, 2H), 1.81 (dd, J=25.2, 10.6 Hz, 2H),1.35-1.14 (m, 2H), 0.99 (t, J=7.4 Hz, 3H). Purity 98%. HRMS calculatedfor C₁₆H₂₁N₃O₄+H⁺ 320.1610. found (ESI(+), [M+H]) 320.1634.

1-(1-Propionylpiperidin-4-yl)-3-(3,4,5-trimethoxyphenyl)urea. (18)

3,4,5-Trimethoxyphenyl isocyanate was prepared from3,4,5-trimethoxyaniline by Method A and was subsequently reacted with 1by Method B. Flash chromatography eluted with 15:1 EtOAc:MeOH andrecrystallization from EtOAc afforded compound 18 (56 mg, 15%) as awhite solid: Mp 173-175° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.28 (s, 1H),6.72 (s, 2H), 6.10 (d, J=7.6 Hz, 1H), 4.18 (d, J=13.3 Hz, 1H), 3.76 (d,J=13.9 Hz, 1H), 3.71 (s, 6H), 3.69-3.63 (m, 1H), 3.58 (s, 3H), 3.11 (t,J=11.4 Hz, 1H), 2.78 (t, J=11.2 Hz, 1H), 2.32 (dd, J=7.4, 7.4 Hz, 2H),1.87-1.75 (m, 2H), 1.35-1.15 (m, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity99%. HRMS calculated for C₁₈H₂₇N₃O₂−H⁺ 364.1873. found (ESI(−), [M−H])364.1906.

1-(4-Morpholinophenyl)-3-(1-propionylpiperidin-4-yl)urea. (19)

4-Morpholinophenyl isocyanate was prepared from 4-morpholinoaniline byMethod A using 1M NaOH in brine as the base and was subsequently reactedwith 1 by Method B. Flash chromatography eluted with 9:1 EtOAc:MeOHafforded compound 19 (61 mg, 17%) as a white solid: Mp 221-225° C. ¹HNMR (500 MHz, DMSO-d₆) δ 8.06 (s, 1H), 7.23 (d, J=8.9 Hz, 2H), 6.83 (d,J=8.9 Hz, 2H), 6.03 (d, J=7.6 Hz, 1H), 4.17 (d, J=13.5 Hz, 1H),3.79-3.73 (obscured d, 1H), 3.71 (t, J=12.2 Hz, 4H), 3.69-3.62 (m, 1H),3.11 (t, J=12.2 Hz, 1H), 2.98 (t, 4H), 2.78 (t, J=11.7 Hz, 1H), 2.31 (q,J=7.4 Hz, 2H), 1.87-1.75 (m, 2H), 1.34-1.13 (m, 2H), 0.98 (t, J=7.4 Hz,3H). Purity 93%. HRMS calculated for C₁₉H₂₈N₄O₃+H⁺ 359.2083. found(ESI(+), [M+H]) 359.2068.

1-(4-Nitrophenyl)-3-(1-propionylpiperidin-4-yl)urea. (20)

4-Nitrophenyl isocyanate was reacted with 1 by Method B. Flashchromatography eluted with 15:1 EtOAc:MeOH afforded compound 20 (122 mg,38%) as a white solid: Mp 240-241° C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.15(s, 1H), 8.14 (d, J=9.2 Hz, 2H), 7.61 (d, J=9.2 Hz, 2H), 6.49 (d, J=7.5Hz, 1H), 4.20 (d, J=13.2 Hz, 1H), 3.80-3.68 (m, 2H), 3.13 (t, J=12.8 Hz,1H), 2.79 (t, J=11.9 Hz, 1H), 2.32 (q, J=7.4 Hz, 2H), 1.83 (dd, J=25.2,12.5 Hz, 2H), 1.39-1.19 (m, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity 95%.HRMS calculated for C₁₅H₂₀N₄O₄−H⁺ 319.1407. found (ESI(−), [M−H])319.1410.

Methyl 4-(3-(1-propionylpiperidin-4-yl)ureido)benzoate. (21)

Methyl 4-isocyanatobenzoate was reacted with 1 by Method B. Flashchromatography eluted with 15:1 EtOAc:MeOH afforded compound 21 (271 mg,82%) as a white solid: Mp 201-204° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.79(s, 1H), 7.83 (d, J=8.8 Hz, 2H), 7.50 (d, J=8.8 Hz, 2H), 6.34 (d, J=7.5Hz, 1H), 4.18 (d, ./=13.0 Hz, 1H), 3.80 (s, 3H), 3.78-3.65 (m, 2H), 3.13(t, J=11.9 Hz, 1H), 2.79 (t, J=11.9 Hz, 1H), 2.32 (q, J=7.4 Hz, 2H),1.83 (dd, J=25.29, 11.74 Hz, 2H), 1.38-1.27 (m, 1H), 1.27-1.16 (m, 1H),0.98 (t, J=7.4 Hz, 3H). Purity 96%. FIRMS calculated for C₁₇H₂₃N₃O₄−H⁺332.1611. found (ESI(−), [M−H]) 332.1595.

4-(3-(1-Propionylpiperidin-4-yl)ureido)benzoic acid. (22)

Compound 21 (85 mg, 0.25 mmol) was refluxed in ethanol (10 mL)containing 1M NaOH (300 μl, 1.2 eq) for 5 hours. Additional base (300μl) was added and the reaction continued for 2 hours before cooling toRT The reaction was quenched with 1N HCl (20 mL), the organic solventremoved and the remaining suspension filtered to give compound 22 (49mg, 60%) as a white solid: MP 201-204° C. ¹H NMR (500 MHz, DMSO-d₆) δ12.50 (s, 1H), 8.73 (s, 1H), 7.80 (d, J=8.7 Hz, 2H), 7.47 (d, J=8.7 Hz,2H), 6.32 (d, J=7.6 Hz, 1H), 3.13 (t, J=12.1 Hz, 1H), 2.80 (t, J=11.8Hz, 1H), 2.32 (q, J=7.4 Hz, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity 94%.HRMS calculated for C₁₆H₂₁N₃O₄−H⁺ 318.1454. found (ESI(−), [M−H])318.1498.

1-(4-Hydroxyphenyl)-3-(1-propionylpiperidin-4-yl)urea. (23)

4-Benzyloxyphenyl isocyanate was prepared from 4-benzyloxyanline byMethod A and was subsequently reacted with 1 by Method B. Flashchromatography eluted with EtOAc gave intermediate1-(4-benzyloxyphenyl)-3-(1-propionylpiperidin-4-yl)urea, which wasdissolved in ethanol and hydrogenolyzed with 10% palladium on charcoalunder an atmosphere of hydrogen. Flash chromatography eluted with 15:1DCM:MeOH gave compound 23 (13 mg, 5% overall) as a white solid: Mp229-230° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.92 (s, 1H), 7.97 (s, 1H), 7.13(d, J=8.6 Hz, 2H), 6.62 (d, J=8.7 Hz, 2H), 6.00-5.97 (m, 1H), 4.17 (d,J=12.5 Hz, 1H), 3.75 (d, J=14.3 Hz, 1H), 3.70-3.62 (m, 1H), 3.11 (t,J=12.1 Hz, 1H), 2.81-2.74 (m, 1H), 2.31 (q, J=7.5 Hz, 2H), 1.80 (dd,J=26.6, 12.1 Hz, 2H), 1.33-1.23 (m, 1H), 1.23-1.13 (m, 1H), 0.98 (t,J=7.4 Hz, 3H). Purity 100%. HRMS calculated for C₁₅H₂₁N₃O₃+H⁺ 292.1661.found (ESI(+), [M+H]) 292.1618.

1-(4-Fluorophenyl)-3-(1-propionylpiperidin-4-yl)urea. (24)

4-Fluorophenyl isocyanate was prepared from 4-fluoroaniline by Method Aand was subsequently reacted with 1 by Method B. Flash chromatographyeluted with EtOAc afforded compound 24 (106 mg, 36%) as a white solid:Mp 183-184° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.36 (s, 1H), 7.40-7.35 (m,2H), 7.08-7.02 (m, 1H), 6.14 (d, J=7.6 Hz, 1H), 4.18 (d, J=13.1 Hz, 1H),3.75 (d, J=13.9 Hz, 1H), 3.72-3.64 (m, 1H), 3.12 (t, J=12.1 Hz, 1H),2.78 (t, J=11.3 Hz, 1H), 2.32 (dd, J=7.4 Hz, 2H), 1.88-1.75 (m, 2H),1.36-1.14 (m, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity 100%. HRMS calculatedfor C₁₅H₂₀FN₃O₂−H⁺ 292.1462. found (ESI(−), [M−H]) 292.1444.

1-(4-Chlorophenyl)-3-(1-propionylpiperidin-4-yl)urea. (25)

4-Chlorophenyl isocyanate was reacted with 1 by Method B. Flashchromatography eluted with 15:1 EtOAc:MeOH and recrystallization fromacetone:hexane afforded compound 25 (53 mg, 17%) as a white solid: Mp225-226° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.54 (s, 1H), 7.41 (d, J=8.8 Hz,2H), 7.25 (d, J=8.8 Hz, 2H), 6.26 (d, J=7.6 Hz, 1H), 4.17 (d, J=13.0 Hz,1H), 3.75 (d, J=13.6 Hz, 1H), 3.72-3.64 (m, 1H), 3.12 (t, J=11.23 Hz,1H), 2.79 (t, J=11.0 Hz, 1H), 2.31 (q, J=7.4 Hz, 2H), 1.88-1.74 (m, 2H),1.37-1.15 (m, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity 96%. HRMS calculatedfor C₁₅H₂₀CIN₃O₂−H⁺ 308.1166. found (ESI(−), [M−H]) 308.1152.

1-(4-Bromophenyl)-3-(1-propionylpiperidin-4-yl)urea. (26)

4-Bromophenyl isocyanate was reacted with 1 by Method B. Flashchromatography eluted with EtOAc afforded compound 26 (201 mg, 57%) as awhite solid: Mp 233-239° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.48 (s, 1H),7.40-7.33 (m, 4H), 6.21 (d, J=7.6 Hz, 1H), 4.18 (d, J=13.0 Hz, 1H), 3.75(d, J=13.8 Hz, 1H), 3.72-3.64 (m, 1H), 3.12 (t, J=12.1 Hz, 1H), 2.78 (t,J=11.8 Hz, 1H), 2.31 (q, J=7.3 Hz, 2H), 1.81 (dd, J=25.9, 12.2 Hz, 2H),1.36-1.15 (m, 2H), 0.98 (t, J=7.4 3H). Purity 94%. HRMS calculated forC₁₅H₂₀BrN₃O₂+H⁺ 354.0817. found (ESI(+), [M+H]) 354.0774.

1-(4-Iodophenyl)-3-(1-propionylpiperidin-4-yl)urea. (27)

4-Iodophenyl isocyanate was prepared on a 2 mmol scale from4-iodoaniline by Method A and was subsequently reacted with 1 by MethodB. Trituration twice from 1:1 EtOAc:MeOH, flash chromatography elutedwith 8:1 EtOAc:MeOH and recrystallization from acetone:MeOH affordedcompound 27 (39 mg, 5%) as a white solid: Mp 246-247° C. ¹H NMR (500MHz, DMSO-d₆) δ 8.46 (s, 1H), 7.52 (d, J=8.7 Hz, 2H), 7.23 (d, J=8.7 Hz,2H), 6.21 (d, 7.6 Hz, 1H), 4.17 (d, J=12.9 Hz, 1H), 3.75 (d, J=14.3 Hz,1H), 3.72-3.63 (m, 1H), 3.16-3.06 (m, 1H), 2.82-2.73 (m, 1H), 2.31 (q,J=7.4 Hz, 2H), 1.87-1.74 (m, 2H), 1.35-1.14 (m, 2H), 0.98 (t, J=7.4 Hz,3H). Purity 95%. HRMS calculated for C₁₅H₂₀IN₃O₂−H⁺ 400.0522. found(ESI(−), [M−H]) 400.0488.

1-(3-Fhiorophenyl)-3-(1-propionylpiperidin-4-yl)urea. (28)

3-Fluorophenyl isocyanate was reacted with 1 by Method B. Flashchromatography eluted with EtOAc afforded compound 28 (231 mg, 79%) as awhite solid: Mp 158-164° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.57 (s, 1H),7.44 (d, JHF=12.2 Hz, 1H), 7.23 (q, J=7.9 Hz, 1H), 7.00 (d, J=8.4 Hz,1H), 6.69 (t, J=8.3 Hz, 1H), 6.25 (d, J=7.5 Hz, 1H), 4.18 (d, J=13.0 Hz,1H), 3.76 (d, J=13.7 Hz, 1H), 3.72-3.64 (m, 1H), 3.12 (t, J=12.2 Hz,1H), 2.78 (t, J=11.9 Hz, 1H), 2.32 (q, J=7.3 Hz, 2H), 1.82 (dd, J=25.8,12.3 Hz, 2H), 1.31 (q, J=11.4 Hz, 1H), 1.21 (q, J=11.1 Hz, 1H), 0.98 (t,J=7.3 Hz, 3H). Purity 97%. HRMS calculated for C₁₅H₂₀FN₃O₂−H⁺ 294.1608.found (ESI(−), [M−H]) 294.1587.

1-(2-Fluorophenyl)-3-(1-propionylpiperidin-4-yl)urea. (29)

The reaction of 2-fluorophenyl isocyanate with 1 in the same manner asfor compound 27 afforded compound 29 (118 mg, 40%) as a white solid: Mp127-130° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.19 (d, J=1.3 Hz, 1H), 8.12(dt, J=8.3, 1.0 Hz, 1H), 7.19-7.14 (m, 1H), 7.07 (t, J=7.8 Hz, 1H),6.94-6.88 (m, 1H), 6.69 (d, J=7.4 Hz, 1H), 4.12 (d, J=13.1 Hz, 1H),3.77-3.66 (m, 2H), 3.15 (t, J=11.5 Hz, 1H), 2.86 (t, J=11.5 Hz, 1H),2.32 (q, J=7.4 Hz, 2H), 1.83 (dd, J=25.9, 11.9 Hz, 2H), 1.34-1.25 (m,1H), 1.25-1.14 (m, 1H), 0.98 (t, J=7.4 Hz, 3H). Purity 94%. HRMScalculated for C₁₅H₂₀FN₃O₂−H⁺ 2924.1608. found (ESI(−), [M−H]) 294.1589.

1-(3-Chlorophenyl)-3-(1-propionylpiperidin-4-yl)urea. (30)

3-Chlorophenyl isocyanate was reacted with 1 by Method B. Flashchromatography eluted with EtOAc afforded compound 30 (116 mg, 38%) as awhite solid: Mp 165-166° C. ¹H NMR (500 MHz, DMSO-de) δ 8.56 (s, 1H),7.66 (t, J=1.9 Hz, 1H), 7.23 (t, J=8.0 Hz, 1H), 7.16 (d, J=8.0 Hz, 1H),6.93 (dq, J=8.0, 1.0 Hz, 1H), 6.26 (d, J=7.6 Hz, 1H), 4.19 (brd, J=13.3Hz, 1H), 3.76 (brd, J=13.3 Hz, 1H), 3.72-3.64 (m, 1H), 3.12 (dd, J=11.4,11.4 Hz, 1H), 2.78 (dd, J=11.4, 11.4 Hz, 1H), 2.32 (q, J=7.4 Hz, 2H),1.84 (d, J=1.84, 1H), 1.79 (d, J=12.5 Hz, 1H), 1.33 (dq, J=11.5, 3.8 Hz,2H), 1.21 (dq, J=11.5, 3.8 Hz, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity 97%.HRMS calculated for C₁₅H₂₀ClN₃O₂−H⁺ 308.1166. found (ESI(−), [M−H])308.1111.

1-(2-Chlorophenyl)-3-(1-propionylpiperidin-4-yl)urea. (31)

2-Chloroaniline (128 mg, 1 mmol) was subject to Method C. Flashchromatography eluted with EtOAc afforded compound 31 (48 mg, 16%) as awhite solid: Mp 150-157° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.16 (d, J=8.2Hz, 1H), 7.91 (s, 1H), 7.38 (d, J=7.9 Hz, 1H), 7.22 (t, J=7.7 Hz, 1H),7.05 (d, J=7.2 Hz, 1H), 6.94 (t, J=7.4 Hz, 1H), 4.12 (d, J=12.2 Hz, 1H),3.79-3.67 (m, 2H), 3.16 (t, J=11.4 Hz, 1H), 2.88 (t, J=11.2 Hz, 1H),2.32 (q, J=7.4 Hz, 2H), 1.85 (dd, J=26.0, 11.4 Hz, 2H), 1.37-1.16 (m,2H), 0.99 (t, J=7.4 Hz, 3H). Purity 92%. HRMS calculated forC₁₅H₂₀ClN₃O₂+H⁺ 310.1322. found (ESI(+), [M+H]) 310.1311.

1-(3,4-Dichlorophenyl)-3-(1-propionylpiperidin-4-yl)urea. (32)

3,4-Dichlorophenyl isocyanate was prepared from 3,4-dichloroaniline byMethod A (using NaOH and brine as the base) and was subsequently reactedwith 1 by Method B. Flash chromatography eluted with 17:1 EtOAc:MeOH andrecrystallization from EtOAc afforded compound 32 (68 mg, 20%) as awhite solid: Mp 198-200° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.67 (s, 1H),7.82 (s, 1H), 7.44 (d, J=8.8 Hz, 1H), 7.23 (d, J=8.5 Hz, 1H), 6.32 (d,J=7.8 Hz, 1H), 4.19 (d, J=13.1 Hz, 1H), 3.76 (d, J=13.8 Hz, 1H),3.73-3.64 (m, 1H), 3.11 (t, J=11.8 Hz, 1H), 2.77 (t, J=12.1 Hz, 1H),2.32 (q, J=7.4 Hz, 2H), 1.87-1.75 (m, 2H), 1.37-1.16 (m, 2H), 0.98 (t,J=7.3 Hz, 3H). Purity 98%. HRMS calculated for C₁₅H₁₉Cl₂N₃O₂+H⁺342.0776. found (ESI(+), [M+H]) 342.0757.

1-(3,5-Dichlorophenyl)-3-(1-propionylpiperidin-4-yl)urea. (33)

3,5-Dichlorophenyl isocyanate was prepared from 3,5-dichloroaniline byMethod A and subsequently reacted with 1 by Method B. Flashchromatography eluted with EtOAc and recrystallization fromEtOAc:acetone afforded compound 33 (22 mg, 6%) as a white solid: Mp196-198° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.74 (s, 1H), 7.46 (d, J=1.5 Hz,2H), 7.07 (t, J=1.5 Hz, 1H), 6.40 (d, J=7.6 Hz, 1H), 4.20 (d, J=13.0 Hz,1H), 3.77 (d, J=13.2 Hz, 1H), 3.73-3.64 (m, 1H), 3.11 (t, J=12.1 Hz,1H), 2.75 (t, J=11.9 Hz, 1H), 2.31 (q, J=7.5 Hz, 2H), 1.81 (dd, J=25.8,12.0 Hz, 2H), 1.37-1.16 (m, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity 100%.HRMS calculated for C₁₅H₁₉Cl₂N₃O₂+H⁺ 344.0932. found (ESI(+), [M+H])344.0897.

1-(2,6-DichIorophenyl)-3-(1-propionylpiperidin-4-yl)urea. (34)

2,6-Dichlorophenyl isocyanate was reacted with 1 by Method B. Flashchromatography eluted with EtOAc afforded compound 34 (229 mg, 67%) as awhite solid: Mp 170-174° C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.87 (s, 1H),7.47 (d, J=8.1 Hz, 2H), 7.25 (t, J=8.1 Hz, 1H), 6.38 (d, J=7.8 Hz, 1H),4.20 (d, J=13.2 Hz, 1H), 3.77 (d, J=13.0 Hz, 1H), 3.70-3.60 (m, 1H),3.15-3.05 (m, 1H), 2.75 (t, J=11.4 Hz, 1H), 2.31 (q, J=7.3 Hz, 2H),1.89-1.75 (m, 2H), 1.38-1.16 (m, 2H), 0.98 (t, J=7.4, 3H). Purity 95%.HRMS calculated for C₁₅H₁₉Cl₂N₃O₂−H⁺ 342.0776. found (ESI(−), [M−H])342.0801.

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(1-propionylpiperidin-4-yl)urea.(35)

4-Chloro-3-(trifluoromethyl)phenyl isocyanate was reacted with 1 byMethod B. Flash chromatography eluted with EtOAc and recrystallizationfrom EtOAc afforded compound 35 (89 mg, 24%) as a white solid: Mp182-184° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.85 (s, 1H), 8.06 (s, 1H), 7.54(s, 2H), 6.37 (d, J=7.6 Hz, 1H), 4.20 (d, J=13.2 Hz, 1H), 3.77 (d, J=Hz,1H), 3.70 (s, 1H), 3.11 (t, J=11.8 Hz, 1H), 2.76 (t, J=11.9 Hz, 1H),2.32 (q, J=7.4 Hz, 2H), 1.88-1.74 (m, 2H), 1.38-1.28 (m, 1H), 1.28-1.17(m, 1H), 0.98 (t, J=7.4 Hz, 3H). Purity 95%. HRMS calculated forC₁₆H₁₉ClF₃N₃O₂+H⁺ 378.1196. found (ESI(+), [M+H]) 378.1198.

1-(1-Propionylpiperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea. (36)

4-Trifluoromethylphenyl isocyanate was reacted with 1 by Method B.Recrystallization from EtOAc:hexanes afforded compound 36 (166 mg, 48%)as a white solid: Mp 224-228° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.78 (s,1H), 7.61-7.54 (m, 4H), 6.33 (d, J=7.5 Hz, 1H), 4.19 (d, J=12.9 Hz, 1H),3.83-3.62 (m, 2H), 3.32 (s, 1H), 3.13 (t, J=11.7 Hz, 1H), 2.79 (t,J=11.8 Hz, 1H), 2.32 (dd, J=14.8, 7.4 Hz, 2H), 1.92-1.72 (m, 1H),1.37-1.15 (m, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity 94%. HRMS calculatedfor C₁₆H₂₀F₃N₃O₂−H⁺ 342.1430. found (ESI(−), [M−H]) 342.1404.

1-(1-Propionylpiperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)urea. (37)

3-Trifluoromethylphenyl isocyanate was reacted with 1 by Method B. Flashchromatography eluted with EtOAc and recrystallization fromEtOAc:hexanes afforded compound 37 (30 mg, 9%) as a white solid: Mp153-154° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.72 (s, 1H), 7.96 (s, 1H),7.50-7.41 (m, 2H), 7.22 (d, J=6.9 Hz, 1H), 6.31 (d, J=7.4 Hz, 1H), 4.20(d, J=13.1 Hz, 1H), 3.80-3.73 (m, 1H), 3.74-3.65 (m, 1H), 3.12 (t,J=11.9 Hz, 1H), 2.77 (t, J=11.6 Hz, 1H), 2.32 (q, J=7.4 Hz, 2H),1.88-1.76 (m, 2H), 1.38-1.18 (m, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity98%. HRMS calculated for C₁₆H₂₀F₃N₃O₂−H⁺ 342.1430. found (ESI(−), [M−H])342.1419.

1-(4-Perfluoroisopropylphenyl)-3-(1-propionylpiperidin-4-yl)urea. (38)

4-Perfluoroisopropylaniline was prepared as described previously (J.Org. Chem. 1996, 61, 3929-3934) and converted to the correspondingisocyanate by Method A using NaOH in brine as the base and wassubsequently reacted with 1 by Method B. Flash chromatography elutedwith EtOAc and recrystallization from EtOAc:hexanes afforded compound 38(43 mg, 10%) as a white solid: Mp 160-164° C. dec. ¹H NMR (500 MHz,DMSO-d₆) δ 8.77 (s, 1H), 7.61 (d, J=8.7 Hz, 2H), 7.50 (d, J=8.6 Hz, 2H),6.32 (d, J=7.4 Hz, 1H), 4.19 (d, J=12.6 Hz, 1H), 3.76 (d, J=14.0 Hz,1H), 3.73-3.66 (m, 1H), 3.13 (t, J=12.3 Hz, 1H), 2.79 (t, J=12.0 Hz,1H), 2.32 (q, J=7.3 Hz, 2H), 1.83 (dd, J=25.7, 13.1 Hz, 2H), 1.37-1.17(m, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity 100%. HRMS calculated forC₁₈H₂₀F₇N₃O₂+H⁺ 444.1522. found (ESI(+), [M+H]) 444.1505.

1-(2-Methyl-4-perfluoroisopropylphenyl)-3-(1-propionylpiperidin-4-yl)urea.(39)

2-Methyl-4-perfluoroisopropylaniline (J. Org. Chem. 1996, 61, 3929-3934)and compound 39 (41 mg, 9%) were prepared as for compound 38: Mp226-229° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.23 (d, J=8.6 Hz, 1H), 7.84 (s,1H), 7.40-7.35 (m, 2H), 6.87 (d, J=7.2 Hz, 1H), 4.16 (d, J=12.8 Hz, 1H),3.80-3.67 (m, 2H), 3.15 (t, J=12.2 Hz, 1H), 2.84 (t, J=11.7 Hz, 1H),2.33 (q, J=7.2 Hz, 2H), 2.26 (s, 3H), 1.86 (dd, J=27.2, 12.2 Hz, 2H),1.36-1.27 (m, 1H), 1.27-1.15 (m, 1H), 0.99 (t, J=7A Hz, 3H). Purity100%. FIRMS calculated for C₁₉H₂₂F₇N₃O₂−H⁺ 456.1522. found (ESI(−),[M−H]) 456.1512.

1-(1-Propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea. (40)

To an ice cold solution of intermediate 41 (242 mg, 0.84 mmol) in DCM (5mL) was added triethylamine (250 μL, 1.8 mmol) followed by propionylchloride (95 μL, 1.1 mmol). The reaction was allowed to warm to rt andwas stirred for 5 hours. Flash chromatography eluted with 15:1EtOAc:MeOH afforded compound 40 (178 mg, 63%) as a white solid: Mp195-196° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.55 (s, 1H), 7.47 (d, J=8.9 Hz,2H), 7.22 (d, J=8.6 Hz, 2H), 6.23 (d, J=7.6 Hz, 1H), 4.18 (d, J=12.8 Hz,1H), 3.76 (d, J=13.6 Hz, 1H), 3.73-3.65 (m, 1H), 3.12 (t, J=11.4 Hz,1H), 2.78 (t, J=11.0 Hz, 1H), 2.32 (q, J=7.4 Hz, 2H), 1.82 (dd, J=25.3,12.1 Hz, 2H), 1.36-1.15 (m, 2H), 0.98 (t, J=7.4 Hz, 3H). Purity 97%.HRMS calculated for C₁₆H₂₀F₃N₃O₃−H⁺ 358.1379. found (ESI(−), [M−H])358.1404.

tert-Butyl4-(3-(4-(trifluoromethoxy)phenyl)ureido)piperidine-1-carboxylate. (41)

4-Trifluoromethoxyphenyl isocyanate (1.03 g, 5 mmol) was dissolved indry THF (10 mL) and cooled in an ice bath. A solution ofN—BOC-4-aminopiperidine (781 mg, 5 mmol) in dry THF (10 mL) was slowlyadded. The reaction was allowed to warm to RT and stir for 12 hours. Thesolvent was removed and the residue chromatographed from ethyl acetateto give intermediate 41 (1.71 g, 95%) as a white solid: Mp 160-162° C.¹H NMR (500 MHz, DMSO-d₆) δ 8.54 (s, 1H), 7.47 (d, J=9.0 Hz, 2H), 7.21(d, J=8.6 Hz, 2H), 6.22 (d, J=7.6 Hz, 1H), 3.81 (d, J=13.0 Hz, 2H),3.67-3.59 (m, 1H), 2.90 (s, 2H), 1.79 (dd, J=12.6, 2.5 Hz, 2H), 1.40 (s,9H), 1.25 (dq, J=12.1, 3.9 Hz, 2H). HRMS calculated for C₁₈H₂₄F₃N₃O₄−H⁺402.1641. found (ESI(−), [M−H]) 402.1612.

1-(Piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea. (42)

Intermediate 41 (2.02 g, 5.0 mmol) was treated with 1M HCl in methanol(35 mL) and refluxed for 3 hours. The solvent was evaporated and theresidue diluted with 1N NaOH. The resulting precipitate was removed byfiltration and further dried under high vacuum to give intermediate 42(1.35 g, 89%) as a white solid: Mp 169-173° C. ¹H NMR (500 MHz, DMSO-d₆)δ 8.49 (s, 1H), 7.46 (d, J=9.0 Hz, 2H), 7.21 (d, J=8.6 Hz, 2H), 6.15 (d,J=7.7 Hz, 1H), 3.55-3.45 (m, 1H), 2.89 (td, J=12.8, 3.5 Hz, 2H),2.49-2.45 (m, 2H), 2.13 (s, 1H), 1.74 (dd, J=12.4, 2.4 Hz, 2H), 1.21(dq, J=11.7, 3.7 Hz, 2H). HRMS calculated for C₁₃H₁₆F₃N₃O₂−H⁺ 302.1117.found (ESI(−), [M−H]) 302.1114.

Propyl 3,4,5-tribenzyloxybenzoate. (43)

Prepared according to J. Med. Chem. 2006, 49, 2829-2837. ¹H NMR (500MHz, CDCl₃) δ 7.43 (d, J=7.2 Hz, 4H), 7.41-7.30 (m, 10H), 7.30-7.23 (m,3H), 5.14 (s, 4H), 5.12 (s, 2H), 4.24 (t, J=6.7 Hz, 2H), 1.81-1.72 (sxt,2H), 1.01 (t, J=7.4 Hz, 3H).

3,4,5-Tribenzyloxybenzoic acid. (44)

Prepared according to J. Med. Chem. 2006, 49, 2829-2837.

Ethyl (4-benzyl-piperazin-1-yl)acetate. (45)

Prepared according to J. Med. Chem. 2003, 46, 1918-1930. ¹H NMR (500MHz, DMSO-d₆, 50° C.) δ 7.33-7.19 (m, 5H), 4.08 (q, J=7.1 Hz, 2H), 3.45(s, 2H), 3.17 (s, 2H), 2.51 (dd, J=4.5, 4.5 Hz, 2H) 2.50-2.45 (m, 2H),2.37 (dd, J=4.5, 4.5 Hz, 4H), 1.18 (t, J=7.1 Hz, 3H).

(4-Benzyl-piperazin-1-yl)acetic acid. (46)

Prepared according to J. Med. Chem. 2003, 46, 1918-1930. ¹H NMR (500MHz, DMSO-d₆) δ 7.32 (tt, J=6.9, 1.4, 2H) 7.29 (d, J=6.9 Hz, 2H) 7.24(t, J=6.9 Hz, 1H), 3.46 (s, 2H), 3.13 (s, 2H), 2.64 (brs, 4H), 2.42(brs, 4H).

1-(1-Isonicotinoylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea(47)

Intermediate 42 (152 mg, 0.5 mmol) was reacted with isonicotinic acid byMethod E. Flash chromatography eluted with 9:1 DCM:MeOH affordedcompound 47 (204 mg, 100%) as a white solid: Mp 210-212° C. ¹H NMR (500MHz, DMSO-d₆) δ 8.67 (d, J=6 Hz, 2H), 8.59 (s, 1H), 7.47 (d, J=9 Hz,2H), 7.38 (d, J=6 Hz, 2H), 7.22 (d, J=9 Hz, 2H), 6.25 (d, J=8 Hz, 1H),4.30 (d, J=12 Hz, 1H), 3.76 (s, 1H), 3.41 (d, J=12 Hz, 1H), 3.16 (t,J=12 Hz, 1H), 3.05 (t, J=12 Hz, 1H), 1.97-1.88 (m, 1H), 1.84-1.75 (m,1H), 1.47-1.29 (m, 2H). Purity 92%. FIRMS calculated for C₁₉H₁₉F₃N₄O₃−H⁺407.1331. found (ESI(−), [M−H]) 407.1316.

1-(1-(6-Chloronicotinoyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.(48)

The reaction of 42 with 6-chloronicotinic in the same manner as forcompound 46 afforded compound 48 (220 mg, 100%) as a white solid: Mp208-209° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.54 (s, 1H), 8.46 (d, J=2 Hz,1H), 7.90 (dd, J=8, 2 Hz, 1H), 7.60 (d, J=8 Hz, 1H), 7.47 (d, J=9 Hz,2H), 7.20 (d, J=9 Hz, 2H), 6.22 (d, J=7 Hz, 1H), 4.28 (s, 1H), 3.76 (s,1H), 3.50 (s, 1H), 3.26-3.02 (m, 2H), 1.87 (s, 2H), 1.40 (s, 2H). Purity94%. HRMS calculated for C₁₉H₁₈ClF₃N₄O₃−H⁺ 441.0942. found (ESI(−),[M−H]) 441.0942.

1-(((Pyridin-2-yl)acetyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.(49)

The reaction of 42 with 2-pyridyl acetic acid in the same manner as forcompound 46 afforded compound 49 (196 mg, 93%) as a white solid: Mp183-187° C. ¹H NMR (500 MHz, DMSO-de) δ 8.48 (d, J=4.3 Hz, 1H), 8.43 (s,1H), 7.73 (dt, J=7.7, 1.7 Hz, 1H), 7.46 (d, J=9.0 Hz, 2H), 7.28 (d,J=7.8 Hz, 1H), 7.26-7.22 (m, 1H), 7.19 (d, J=8.6 Hz, 2H), 6.17 (d, J=7.5Hz, 1H), 4.16 (d, J=12.0 Hz, 1H), 3.91 (d, J=13.3 Hz, 1H), 3.87 (s, 2H),3.74-3.65 (m, 1H), 3.20-3.14 (m, 1H), 2.86 (t, J=11.9 Hz, 1H), 1.84-1.75(m, 2H), 1.27-1.15 (m, 2H). Purity 91%. HRMS calculated forC₂₀H₂₁F₃N₄O₃−H⁺ 421.1488. found (ESI(−), [M−H]) 421.1477.

1-(1-(2-(4-Benzylpiperazin-1-yl)acetyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.(50)

Intermediate 42 (303 mg, 1 mmol) was reacted with carboxylic acid 46 byMethod E. Flash chromatography eluted with 9:1 DCM:MeOH affordedcompound 50 (379 mg, 73%) as a white solid: Mp 178-183° C. ¹H NMR (500MHz, DMSO-d₆) δ 8.52 (s, 1H), 7.47 (d, J=8.9 Hz, 2H), 7.34-7.26 (m, 4H),7.26-7.19 (m, 3H), 6.25 (d, J=7.5 Hz, 1H), 4.14 (d, J=13.4 Hz, 1H), 3.95(d, J=12.5 Hz, 1H), 3.75-3.66 (m, 1H), 3.45 (s, 2H), 3.23 (d, J=13.2 Hz,1H), 3.12 (t, J=11.9 Hz, 1H), 3.00 (d, J=13.1 Hz, 1H), 2.78 (t, J=11.6Hz, 1H), 2.47-2.30 (m, 8H), 1.89-1.76 (m, 2H), 1.40-1.31 (m, 1H),1.23-1.13 (m, 1H). Purity 90%. FIRMS calculated for C₂₀H₂₁F₃N₄O₃−H⁺518.2379. found (ESI(−), [M−H]) 518.2365.

1-(1-((4-Acetylpiperazin-1-yl)acetyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.(51)

Compound 50 (110 mg, 0.21 mmol) was deprotected by stirring overnightwith 10% Pd/C in ethanol (15 mL) under a hydrogen atmosphere. Thereaction was filtered through a bed of celite and the filtrateevaporated to give intermediate1-(1-((piperazin-1-yl)acetyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea(103 mg, quantitative) as a clear oil, which was used withoutpurification and coupled with acetic acid by Method E. Flashchromatography eluted with 9:1 DCM:MeOH and recrystallization fromEtOAc:hexanes afforded compound 51 (73 mg, 74% over 2 steps) as a whitesolid: Mp 111-122° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.52 (s, 1H), 7.47 (d,J=8.9 Hz, 2H), 7.22 (d, J=Hz, 2H), 6.25 (d, J=7.7 Hz, 1H), 4.15 (d,J=13.1 Hz, 1H), 3.92 (d, J=13.2 Hz, 1H), 3.76-3.67 (m, 1H), 3.47-3.38(m, 4H), 3.25 (d, J=13.5 Hz, 1H), 3.12 (q, J-13.0 Hz, 2H), 2.81 (t,J=11.5 Hz, 1H), 2.37-2.32 (m, 2H), 1.98 (s, 3H), 2.44-2.40 (m, 2H),1.90-1.77 (m, 2H), 1.42-1.31 (m, 1H), 1.26-1.15 (m, 1H). Purity 90%.HRMS calculated for C₂₀H₂₁F₃N₄O₃−H⁺ 470.2015. found (ESI(−), [M−H])470.2009.

1-(1-(Cyclopropanecarbonyl)piperidin-4-yI)-3-(4-(trifluoromethoxy)phenyl)urea.(52)

Intermediate 42 (76 mg, 0.25 mmol) was reacted with cyclopropanecarboxylic acid by Method E. Flash chromatography eluted with EtOAc andrecrystallization from EtOAc:hexane afforded compound 52 (47 mg, 51%) asa white solid: Mp 195-196° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.56 (s, 1H),7.47 (d, J=9.0 Hz, 2H), 7.22 (d, J=8.7 Hz, 2H), 6.25 (d, J=7.7 Hz, 1H),4.15 (t, J=14.8 Hz, 2H), 3.77-3.68 (m, 1H), 3.25 (t, J=11.5 Hz, 1H,obscured), 2.81 (t, J=11.1 Hz, 1H), 1.98 (m, 1H), 1.93-1.75 (m, 2H),1.41-1.17 (m, 2H), 0.75-0.65 (m, 4H). Purity 100%. HRMS calculated forC₁₇H₂₀F₃N₃O₃+H⁺ 372.1535. found (ESI(+), [M+H]) 372.1546.

1-(1-(Trifluoroacetyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.(53)

Intermediate 42 (100 mg, 0.33 mmol) was dissolved in dry THF (1 mL),ethyl trifluoroacetate (30 μl, 0.39 mmol) was added and the reaction wasrefluxed for 18 hours. The reaction was cooled to RT, evaporated and theresidue chromatographed from EtOAc to give compound 53 (49 mg, 37%) as awhite solid: Mp 150-154° C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.59 (s, 1H),7.47 (d, J=9.0 Hz, 2H), 7.22 (d, J=8.6 Hz, 2H), 6.27 (d, J=1.6 Hz, 1H),4.15 (d, J=13.1 Hz, 1H), 3.80 (d, J=11.1 Hz, 2H), 3.38 (t, J=11.9 Hz,1H), 3.11 (t, J=12.1 Hz, 1H), 1.94 (t, J=15.2 Hz, 2H), 1.46-1.34 (m,2H). Purity 100%. HRMS calculated for C₁₅H₁₅F₆N₃O₃+H⁺ 400.1096. found(ESI(+), [M+H]) 400.1078.

1-(4-(Trifhioromethoxy)phenyl)-3-(1-(3,4,5-trihydroxybenzoyl)piperidin-4-yl)urea.(54)

Intermediate 42 (152 mg, 0.5 mmol) was reacted with 44 by Method E. Thereaction was diluted in ethyl acetate and washed with 1M NaOH, 1N HCland finally water. The organic phase was dried and evaporated and theresidue recrystallized from EtOAc:acetone to give intermediate1-(4-(trifluoromethoxy)phenyl)-3-(1-(3,4,5-tris(benzyloxy)benzoyl)piperidin-4-yl)urea(304 mg, 84%). This intermediate (145 mg, 0.20 mmol) was hydrogenated inthe same manner as for compound 51 and recrystallized from EtOAc:acetoneto afford compound 54 (51 mg, 47% over 2 steps) as a white solid: Mp168-175° C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.07 (s, 2H), 8.56 (s, 1H), 8.45(s, 1H), 7.47 (d, J=9.0 Hz, 2H), 7.21 (d, J=9.0 Hz, 2H), 6.31 (s, 2H),6.26 (d, J=7.6 Hz, 1H), 3.73 (s, 1H), 3.03 (s, 2H), 1.83 (d, J=9.6 Hz,2H), 1.31 (dd, J=22.0, 11.9 Hz, 2H). Purity 94%. HRMS calculated forC₂₀H₂₀F₃N₃O₆−H⁺ 454.1226. found (ESI(−), [M−H]) 454.1241.

1-(1-(Methylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.(55)

Intermediate 42 (152 mg, 0.5 mmol) was reacted with methanesulfonylchloride by Method E. Recrystallization from EtOAc:acetone affordedcompound 55 (160 mg, 84%) as a white solid: Mp 233-234° C. ¹H NMR (500MHz, DMSO-d₆) δ 8.57 (s, 1H), 7.47 (d, J=9.0 Hz, 2H), 7.22 (d, J=8.6 Hz,2H), 6.29 (d, J=7.5 Hz, 1H), 3.64-3.55 (m, 1H), 3.47 (d, J=12.2 Hz, 2H),2.91-2.84 (m, 5H), 1.94-1.88 (m, 2H), 1.50-1.40 (m, 2H). Purity 98%.HRMS calculated for C₁₄H₁₈F₃N₃O₄S−H⁺ 380.0892. found (ESI(−), [M−H])380.0931.

1-(1-(Ethylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.(56)

The reaction of 42 with ethanesulfonyl chloride in the same manner asfor compound 55 afforded compound 56 (122 mg, 62%): Mp 235-239° C. ¹HNMR (500 MHz, DMSO-d₆) δ 8.56 (s, 1H), 7.47 (d, J=9.1 Hz, 2H), 7.22 (d,J=8.7 Hz, 2H), 6.29 (d, J=7.6 Hz, 1H), 3.66-3.58 (m, 1H), 3.52 (d,J=12.6 Hz, 2H), 3.05 (q, J=7.3 Hz, 2H), 3.00-2.93 (m, 2H), 1.89 (dd,J=12.8 Hz, 2H), 1.41 (dq, J=11.5, 4.0 Hz, 2H), 1.21 (t, J=7.3 Hz, 3H).Purity 100%). HRMS calculated for C₁₅H₂₀F₃N₃O₄S+H⁺ 396.1205. found(ESI(+), [M+H]) 396.1179.

1-(1-(Phenylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.(57)

The reaction of 42 with benzenesulfonyl chloride in the same manner asfor compound 55 afforded compound 57 (183 mg, 82%): Mp 188-189° C. ¹HNMR (500 MHz, DMSO-d₆) δ 8.45 (s, 1H), 7.80-7.61 (m, 4H), 7.41 (d, J=9.4Hz, 2H), 7.19 (d, J=9.4 Hz, 2H), 6.21 (d, J=7.5 Hz, 1H), 3.55-3.39 (m,3H), 2.54 (t, J=10.5 Hz, 2H), 1.92-1.80 (m, 2H), 1.35-1.53 (m, 2H).Purity 95%. HRMS calculated for C₁₉H₂₀F₃N₃O₄S−H⁺ 442.1049. found(ESI(−), [M−H]) 442.1046.

1-(1-Tosylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea. (58)

The reaction of 42 with tosyl chloride in the same manner as forcompound 55 afforded compound 58 (119 mg, 52%): Mp 205-207° C. ¹H NMR(500 MHz, DMSO-d₆) δ 8.44 (s, 1H), 7.64 (d, J=8. Hz, 2H), 7.44 (dd, J=8.Hz, 4H), 7.18 (d, J=8. Hz, 2H), 6.18 (d, J=7. Hz, 1H), 3.52-3.45 (m,1H), 3.41 (d, J=12. Hz, 2H), 2.56 (t, J=11. Hz, 2H), 2.42 (s, 3H), 1.87(d, J=12. Hz, 2H), 1.45 (dd, J=10. Hz, 2H). Purity 95%. HRMS calculatedfor C₂₀H₂₂F₃N₃O₄S−H⁺ 456.1205. found (ESI(−), [M−H]) 456.1247.

1-(1-(5-(Dimethylamino)naphthalen-1-ylsuIfonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.(59)

Intermediate 42 (200 mg, 0.7 mmoJ) was dissolved in DCM (7 mL) andtriethylamine (100 μL, 0.7 mmol) added followed by5-(dimethylamino)-1-naphthalenesulfonyl chloride (207 mg, 0.77 mmol).The reaction was evaporated, reconstituted in EtOAc and washed with 1NHCl and 1N K₂CO₃. Flash chromatography eluted with 2:1 EtOAc:hexanesafforded compound 59 (338 mg, 93%) as a tan solid: Mp 102-107° C. ¹H NMR(500 MHz, DMSO-d₆) δ 8.52 (d, J=8.5 Hz, 1H), 8.45 (s, 1H), 8.28 (d,J=8.7 Hz, 1H), 8.14 (d, J=7.3 Hz, 1H), 7.67 (t, J=7.5 Hz, 1H), 7.62 (t,J=8.1 Hz, 1H), 7.43 (d, J=9.1 Hz, 2H), 7.28 (d, J=7.5 Hz, 1H), 7.19 (d,J=8.7 Hz, 2H), 6.24 (d, J=7.5 Hz, 1H), 3.60-3.53 (m, 3H), 2.88 (t,J=10.5 Hz, 2H, obscured by s δ 2.84), 2.84 (s, 6H), 1.84 (dd, J=12.9,3.0 Hz, 2H), 1.38 (dq, J=10.6, 3.6 Hz, 2H). Purity 97%. HRMS calculatedfor C₂₅H₂₇F₃N₄O₄S+H⁺ 537.1783. found (ESI(+), [M+H]) 537.1785.

Example 2 Synthesis of1-(piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea (PTU)

Synthesis of tert-butyl4-(3-(4-(trifluoromethyl)phenyl)ureido)piperidine-1-carboxylate.4-(Trifluoromethyl)phenyl isocyanate (1.068 g, 5.71 mmol) and4-amino-1-Boc-piperidine (1.0 g, 5 mmol) was dissolved in THF (100 mL)and stirred for 12 h. The reaction was quenched by addition of water.The organic layer was isolated and the aqueous layer was extracted withEtOAc for 4 times. The combined organic layer was concentrated undervacuo and was further purified by flash chromatography (EtOAc:Hex/1:1)yielding final product (1.7 g, 4.39 mmol, 88%). ¹H NMR (d₆-DMSO, 300Mhz): δ 8.77 (s, 1H), 7.55 (s, 4H), 6.26 (d, J=10 Hz, 1H), 3.80 (d,J=Hz, 2H), 3.64 (m, 1H), 2.88 (br, 2H), 1.78 (d, J=5 Hz, 2H), 1.38 (s,9H), 1.23 (m, 2H).

Synthesis of 1-(piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea (PTU).tert-butyl4-(3-(4-(trifluoromethyl)phenyl)ureido)piperidine-1-carboxylate (1.6 g,4.13 mmol) was dissolved in HCl solution (2M, MeOH, 100 mL). Theresulting solution was refluxed for 2 h. The solvent was removed undervacuo and the crude was basified by NaOH solution (2N). The finalprecipitates (0.9 g, 3.13 mmol, 78%) were filtered and dried under highvacuum. The final product (PTU) was served as a scaffold for thefollowing urea inhibitors synthesis. ¹H NMR (d₆-DMSO, 300 Mhz): δ 8.77(s, 1H), 7.57 (s, 4H), 6.30 (d, J=7.8 Hz, 1H), 3.55 (m, 1H), 2.91 (d,J=12.6 Hz, 2H), 2.48 (d, J=11.1 Hz, 2H), 1.77 (d, J=11 Hz, 2H), 1.22 (m,2H).

Example 3 Synthesis of1-(1-isobutyrylpiperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea. (60)

Isobutyric acid (37 mg, 0.418 mmol), DMAP (51 mg, 0.418 mmol) and EDCI(59 mg, 0.334 mmol) was dissolved in DMF (10 mL). PTU (80 mg, 0.278mmol) was dissolved in DMF (5 mL) and was added into the reactionmixture dropwisely. The reaction mixture was stirred for 12 h and wasquenched by addition of HCl solution (1M). The organic layer wascollected and the aqueous layer was extracted with EtOAc for 4 times.The combined organic layer was concentrated under vacuo and furtherpurified by flash chromatography (EtOAc:Hex/1:1) yielding final product(90 mg, 0.252 mmol, 90% yield). ¹H NMR (d₆-DMSO, 300 Mhz): δ 8.79 (s,1H), 7.57 (s, 4H), 6.38 (d, J=7.8 Hz, 1H), 4.25 (d, J=Hz, 1H), 3.85 (d,J=12.6 Hz, 1H), 3.75 (m, 1H), 3.18 (t, J=Hz, 1H), 2.85 (m, J=Hz, 1H),2.79 (t, J=Hz, 1H), 1.85 (t, J=Hz, 2H), 1.25 (m, J=Hz, 2H), 0.98 (s,6H).

Example 4 Synthesis of1-(1-cyclopropanecarbonylpiperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.(61)

Cyclopropanecarboxylic acid (36 mg, 0.418 mmol) was reacted with PTU(100 mg, 0.348 mmol) as the same manner as the synthesis of1-(1-isobutyrylpiperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (110 mg, 0.310 mmol, 89% yield). ¹H NMR(d₆-DMSO, 300 Mhz): δ 8.80 (s, 1H), 7.57 (s, 4H), 6.37 (d, J=12 Hz, 1H),4.16 (br, 2H), 3.74 (m, 1H), 3.25 (t, J=11.7 Hz, 1H), 2.81 (t, J=10.8Hz, 1H), 1.98 (m, 1H), 1.85 (m, 2H), 1.30 (m, 2H), 0.7 (s, 4H).

Example 5 Synthesis of1-(1-(3,3,3-trifluoropropionyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.(62)

3,3,3-Trifluoropropionic acid (67 mg, 0.522 mmol) was reacted with PTU(100 mg, 0.348 mmol) as the same manner as the synthesis of1-(1-isobutyrylpiperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (120 mg, 0.302 mmol, 87% yield). ¹H NMR(d₆-DMSO, 300 Mhz): δ 8.80 (s, 1H), 7.58 (s, 4H), 6.38 (d, J=7.8 Hz,1H), 4.19 (d, J=13.3 Hz, 1H), 3.79 (d, J=13.8 Hz, 1H), 3.71 (m, 4H),3.16 (t, J=11.1 Hz, 1H), 2.85 (t, J=11.7 Hz, 1H), 1.84 (m, 2H), 1.30 (m,2H).

Example 6 Synthesis of1-(1-butyrylpiperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea. (63)

Butyric acid (37 mg, 0.418 mmol) was reacted with PTU (80 mg, 0.278mmol) as the same manner as the synthesis of1-(1-isobutyrylpiperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (91 mg, 0.255 mmol, 91% yield). SH NMR(d₆-DMSO, 300 Mhz): δ 8.79 (s, 1H), 7.56 (s, 4H), 6.36 (d, J=7.8 Hz,1H), 4.19 (d, J=12.9 Hz, 1H), 3.75 (d, J=14.4 Hz, 1H), 3.12 (t, J=11.4Hz, 1H), 2.77 (m, J=12 Hz, 1H), 2.28 (t, J=7.5 Hz, 2H), 1.82 (t, J=13.2Hz, 2H), 1.49 (m, 2H), 1.29 (m, 3H), 0.88 (t, J=7.5 Hz, 3H).

Example 7 Synthesis of1-(1-(2-methylbutyryl)piperidin-4-yl)(trifluoromethyl)phenyl)urea. (64)

2-Methylbutyric acid (50 mg, 0.487 mmol) was reacted with PTU (70 mg,0.244 mmol) as the same manner as the synthesis of1-(1-isobutyrylpiperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (80 mg, 0.215 mmol, 88%) yield). ¹H NMR(d₆-DMSO, 300 Mhz): δ 8.77 (d, J=8.4 Hz, 1H), 7.57 (s, 4H), 6.37 (s,1H), 4.22 (m, 1H), 3.88 (d, J=13.2 Hz, 1H), 3.71 (m, 1H), 3.17 (t,J=12.8 Hz, 1H), 2.84 (m, 2H), 1.85 (m, 2H), 1.54 (m, 1H), 1.29 (m, 3H),0.97 (s, 3H), 0.81 (d, J=6 Hz, 3H).

Example 8 Synthesis of1-(1-(4,4,4-trifluorobutyryl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.(65)

4,4,4-Trifluorobutanoic acid (59 mg, 0.418 mmol) was reacted with PTU(80 mg, 0.278 mmol) as the same manner as the synthesis of1-(1-isobutyrylpiperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (80 mg, 0.194 mmol, 70% yield). ¹H NMR(d₆-DMSO, 300 Mhz): δ 8.80 (s, 1H), 7.58 (s, 4H), 6.38 (d, J=7.8 Hz,1H), 4.19 (d, J=13.8 Hz, 1H), 3.79 (d, J=13.8 Hz, 1H), 3.77 (m, 1H),3.16 (t, J=12.3 Hz, 1H), 2.84 (m, J=11.4 Hz, 1H), 2.60 (d, J=6.3 Hz,2H), 2.59 (m, 2H), 1.85 (br, 2H), 1.30 (m, 2H).

Example 9 Synthesis of1-(1-(methylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.(66)

PTU (70 mg, 0.244 mmol) and Et₃N (30 mg, 0.292 mmol) was dissolved inDMF (10 mL) at 0° C. and methylsulfonyl chloride (56 mg, 0.487 mmol) wasadded into the reaction mixture dropwisely. The reaction mixture wasstirred for 12 h at rt and was quenched by addition of HCl solution(1M). The organic layer was collected and the aqueous layer wasextracted with EtOAc for 4 times. The combined organic layer wasconcentrated under vacuo and further purified by flash chromatography(EtOAc:Hex/6:4) yielding final product (45 mg, 0.123 mmol, 51% yield).¹H NMR (d₆-DMSO, 300 Mhz): δ 8.82 (s, 1H), 7.57 (s, 4H), 6.39 (d, J=7.5Hz, 1H), 3.61 (m, 1H), 3.46 (d, J=12.3 Hz, 2H), 2.87 (s, 3H), 2.87 (m,2H), 1.92 (d, J=9.9 Hz, 2H), 1.46 (m, 2H).

Example 10 Synthesis of1-(1-(ethylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.(67)

Ethylsulfonyl chloride (63 mg, 0.487 mmol) was reacted with PTU (70 mg,0.244 mmol) as the same manner as the synthesis of1-(1-(methylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (40 mg, 0.105 mmol, 43% yield). ¹H NMR(d₆-DMSO, 300 Mhz): δ 8.77 (s, 1H), 7.57 (s, 4H), 6.38 (d, J=7.2 Hz,1H), 3.63 (m, 1H), 3.52 (d, J=12.3 Hz, 2H), 3.05 (m, 5H), 1.89 (d, J=10Hz, 2H), 1.47 (m, 2H), 1.22 (t, J=4.8 Hz, 3H).

Example 11 Synthesis of1-(1-(propylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.(69)

Propylsulfonyl chloride (70 mg, 0.487 mmol) was reacted with PTU (70 mg,0.244 mmol) as the same manner as the synthesis of1-(1-(methylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (60 mg, 0.153 mmol, 63% yield). ¹H NMR(d₆-DMSO, 300 Mhz): δ 8.77 (s, 1H), 7.57 (s, 4H), 6.38 (d, J=7.5 Hz,1H), 3.63 (m, 1H), 3.52 (d, J=12.4 Hz, 2H), 3.08 (m, 4H), 1.90 (d,J=9.6, 2H), 1.70 (m, 1H), 1.43 (m, 2H), 0.99 (t, J=7.2 Hz, 3H).

Example 12 Synthesis of1-(1-(butylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.(69)

Butylsulfonyl chloride (76 mg, 0.487 mmol) was reacted with PTU (70 mg,0.244 mmol) as the same manner as the synthesis of1-(1-(methylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (65 mg, 0.160 mmol, 66% yield). ¹H NMR(d₆-DMSO, 300 Mhz): δ 8.78 (s, 1H), 7.57 (s, 4H), 6.38 (d, J=7.5 Hz,1H), 4.19 (d, J=Hz, 1H), 3.82 (d, J=Hz, 1H), 3.73 (m, 1H), 3.18 (t,J=12.4 Hz, 1H), 2.82 (m, 1H), 2.78 (t, J=Hz, 1H), 1.80 (t, J=9.6, 2H),1.43 (m, 2H), 0.97 (s, 6H).

Example 13 Synthesis of1-(1-(cyclopropylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.(70)

Cyclopropylsulfonyl chloride (68.5 mg, 0.487 mmol) was reacted with PTU(70 mg, 0.244 mmol) as the same manner as the synthesis of1-(1-(methylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (65 mg, 0.166 mmol, 68% yield). ¹H NMR(d₆-DMSO, 300 Mhz): δ 8.78 (s, 1H), 7.57 (s, 4H), 6.38 (d, J=7.5 Hz,1H), 4.19 (d, J=Hz, 1H), 3.52 (d, J=12.3 Hz, 2H), 2.99 (t, J=11.4 Hz,2H), 2.58 (m, 1H), 1.91 (d, J=12.6 Hz, 2H), 1.46 (m, 2H), 0.96 (m, 4H).

Example 14 Synthesis of1-(1-(isopropylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.(71)

Isopropylsulfonyl chloride (70 mg, 0.487 mmol) was reacted with PTU (70mg, 0.244 mmol) as the same manner as the synthesis of1-(1-(methylsuIfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (80 mg, 0.203 mmol, 83% yield). ¹H NMR(d₆-DMSO, 300 Mhz): δ 8.78 (s, 1H), 7.57 (s, 4H), 6.38 (d, J=7.5 Hz,1H), 4.19 (d, J=Hz, 1H), 3.82 (d, J=Hz, 1H), 3.73 (m, 1H), 3.18 (t,J=12.4 Hz, 1H), 2.82 (m, 1H), 2.78 (t, J=Hz, 1H), 1.80 (t, J=9.6, 2H),1.43 (m, 2H), 0.97 (s, 6H).

Example 15 Synthesis of1-(1-(2,2,2-trifluoroethylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.(72)

2,2,2-trifluoroethylsulfonyl chloride (89 mg, 0.487 mmol) was reactedwith PTU (70 mg, 0.244 mmol) as the same manner as the synthesis of1-(1-(methylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (48 mg, 0.111 mmol, 46% yield). ¹H NMR(d₆-DMSO, 300 Mhz): δ 8.81 (s, 1H), 7.56 (s, 4H), 6.41 (d, J=6.9 Hz,1H), 4.51 (q, J=9.9 Hz, 2H), 3.58 (d, J=12 Hz, 1H), 3.58 (m, 1H), 3.02(t, J=11.4 Hz, 2H), 1.92 (d, J=12.4 Hz, 2H), 1.45 (m, 2H).

Example 16 Synthesis of1-(1-(3,3,3-trifluoropropylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.(73)

3,3,3-Trifluoropropylsulfonyl chloride (96 mg, 0.487 mmol) was reactedwith PTU (70 mg, 0.244 mmol) as the same manner as the synthesis of1-(1-(methylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (55 mg, 0.123 mmol, 51% yield). ¹H NMR(d₆-DMSO, 300 Mhz): δ 8.78 (s, 1H), 7.57 (s, 4H), 6.38 (d, J=7.5 Hz,1H), 4.19 (d, J=Hz, 1H), 3.82 (d, J=Hz, 1H), 3.73 (m, 1H), 3.18 (t,J=12.4 Hz, 1H), 2.82 (m, 1H), 2.78 (t, J=Hz, 1H), 1.80 (t, J=9.6, 2H),1.43 (m, 2H), 0.97 (s, 6H).

Example 17 Synthesis of1-(1-(cyclopropylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.(74)

Cyclopropylsulfonyl chloride (75 mg, 0.536 mmol) was reacted with ITU(70 mg, 0.268 mmol) as the same manner as the synthesis of1-(1-(methylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureayielding the final product (53 mg, 0.145 mmol, 54.1% yield). ¹H NMR(d₆-DMSO, 300 Mhz): δ 8.20 (s, 1H), 7.26 (d, J=6.9 Hz, 2H), 7.07 (d,J=7.5 Hz, 2H), 6.14 (d, J=7.8 Hz, 1H), 3.59 (m, 1H), 3.52 (d, J=12.8 Hz,2H), 2.98 (t, J=11.4 Hz, 2H), 2.79 (m, 1H), 2.57 (m, 1H), 1.90 (d,J=12.3 Hz, 2H), 1.42 (m, 2H), 1.15 (d, J=6.6 Hz, 6H), 0.95 (m, 4H).

Example 18 Pharmacokinetic Study

Enzyme Purification.

Recombinant murine and human sEH were produced in a polyhedron positivebaculovirus expression system, and were purified by affinitychromatography as previously reported (Arch. Biochem. Biophys. 1993,305, 197-201; J. Biol. Chem. 1993, 268, 17628-17633; Anal. Biochem.1988, 169, 71-80).

IC₅₀ Assay Conditions.

IC₅₀ values were determined using a sensitive fluorescent based assay(Anal. Biochem. 2005, 343, 66-75). Cyano(2-methoxynaphthalen-6-yl)methyltrans-(3-phenyl-oxyran-2-yl) methyl carbonate (CMNPC) was used as thefluorescent substrate. Human sEH (1 nM) or murine sEH (1 nM) wasincubated with the inhibitor for 5 min in pH 7.0 Bis-Tris/HCl buffer (25mM) containing 0.1 mg/mL of BSA at 30° C. prior to substrateintroduction ([S]=5 μM). Activity was determined by monitoring theappearance of 6-methoxy-2-naphthaldehyde over 10 minutes by fluorescencedetection with an excitation wavelength of 330 nm and an emissionwavelength of 465 nm. Reported IC₅₀ values are the average of threereplicates. The fluorescent assay as performed here has a standard errorbetween 10 and 20%, suggesting that differences of two-fold or greaterare significant (Anal. Biochem. 2005, 343, 66-75).

Pharmacokinetics (PK) Study.

Male CFW mice (7 week old, 24-30 g) were purchased from Charles RiverLaboratories. All the experiments were performed according to protocolsapproved by the Animal Use and Care Committee of University ofCalifornia, Davis. Inhibitors (1 mg each) were dissolved in 1 mL ofoleic acid-rich triglyceride containing 20% polyethylene glycol (averagemolecular weight: 400) to give a clear solution for oral administration.Cassette 1 contained compounds 2, 24, 25, and 27. Cassette 2 containedcompounds 12, 13, and 14. Cassette 3 contained compounds 3, 4 and AUDA.Cassette 4 contained compounds 2, 15, and 35. Each cassette was orallyadministered to 3 or 4 mice at a dose of 5 mg/kg in 120-150 μl ofvehicle depending on animal weight. Blood (10 μL) was collected from thetail vein using a pipette tip rinsed with 7.5% EDTA(K3) at 0, 0.5, 1,1.5, 2, 4, 6, 8, 24 hours after oral dosing with the inhibitor. Theblood samples were prepared according to the methods detailed in ourprevious study (Br. J. Pharmacol. 2009, 156, 284-296). Blood sampleswere analyzed using an Agilent 1200 Series HPLC equipped with a 4.6mm×150 mm Inertsil ODS-4 3 μm column (GL Science Inc., Japan) held at40° C. and coupled with an Applied Biosystems 4000 QTRAP hybrid,triple-quadrupole mass spectrometer. The instrument was equipped with alinear ion trap and a Turbo V ion source and was operated in positiveion MRM mode (see Table 6). The solvent system consisted of water/aceticacid (999/1 v/v, solvent A) and acetonitrile/acetic acid (999/1 v/v;solvent B). The gradient was begun at 30% solvent B and was linearlyincreased to 100% solvent B in 5 min. This was maintained for 3 min,then returned to 30% solvent B in 2 min. The flow rate was 0.4 mL/min.The injection volume was 10 μL and the samples were kept at 4° C. in theauto sampler.

There is less than 5% variation in compound levels in replicate bloodsamples from the same mice. Thus the standard deviation shown in FigureS2 represents variation among mice treated with the same compound. ThePK parameters of individual mice were calculated by fitting the timedependent curve of blood inhibitor concentration (Figure S2) to anon-compartmental analysis with the WinNonlin software (Pharsight,Mountain View, Calif.). Parameters determined include the time ofmaximum concentration (T_(max)), maximum concentration (C_(max)),half-life (t_(1/2)), and area under the concentration-time curve toterminal time (AUC_(t)). In separate studies to determine dose linearityof selected compounds, pharmacokinetic parameters determined by cassettedosing were found to be predictive of results from dosing individualcompounds (Br. J. Pharmacol. 2009, 156, 284-296; Anal. Chim. Acta. 2006,559, 37-44).

TABLE 1 Alkyl, Carbocycle and Unsubstituted Aryl Analogues

IC₅₀ (nM)^(a) log P Compd R Human Murine (±0.5) 2

2.8 1.2 3.1 3

3.9 0.9 2.3 4

12 3.5 1.8 5

3.2 0.4 3.5 6

2.7 7.4 1.8 7

130 49 1.3 8

3.0 4.2 2.4 9

3,800 >5,000 nd^(b) ^(a)IC₅₀ values were determined with a fluorescentassay using homogenous recombinant murine and human enzymes (seemethods). ^(b)HPLC method used is limited to log P values greater thanzero.

TABLE 2 Substituted Phenyl Analogues

IC₅₀ (nM) log P Compd R Human Murine (±0.5) 10

1,700 >5,000 1.6 11

40 8.7 1.8 12

43 55 1.8 13

8.3 1.3 2.3 14

2.8 3.3 2.8 15

87 8.7 1.0 16

3.5 0.4 2.8 17

61 100 1.1 18

>5,000 >5,000 0.8 19

2,000 650 0.2 20

38 97 1.7 21

140 64 1.6 22

330 1,000 0.4 23

406 1,400 0.0

TABLE 3 Halophenyl Urea Analogues

IC₅₀ (nM) log P Compd R Human Murine (±0.5) 24

79 110 1.4 25

10 23 2.2 26

3.6 15 2.4 27

7.2 1.4 2.5 28

39 20 1.7 29

300 780 1.6 30

21 6.6 2.2 31

1100 2900 2.0 32

3.4 0.6 2.9 33

0.4 1.0 3.3 34

>5,000 >5,000 1.3 35

4.1 2.3 3.0 36

0.7 6.5 2.4 37

17 8.8 2.4 38

0.4 0.7 3.5 39

17 28 3.8 40

3.7 2.8 2.5

TABLE 4 N-Acyl and N-Sulfonyl Piperidine Analogues

IC₅₀ (nM) log P Compd R² Human Murine (±0.5) 47

0.7 1.3 2.4 48

0.6 0.7 2.9 49

3.1 5.0 2.6 50

1.5 18 3.8 51

0.5 1.2 2.4 52

0.4 0.4 2.7 53

0.4 0.4 3.1 54

0.5 2.7 2.0 55

2.9 2.0 2.2 56

0.4 0.7 2.6 57

1.8 0.4 3.1 58

0.4 0.4 3.5 59

0.8 nd^(a) 4.3 ^(a)nd = Not determined

TABLE 5 Pharmacokinetic Screening Results^(a) C_(max) T_(max) t_(1/2)AUC_(t) Compound (nM) (min) (min) (×10⁴ nM · min) AUDA 14 80 126 0.3 2138 45 78 1.9 3 2,770 60 50 35 4 4,600 50 56 58 12 5,570 30 72 92 134,810 30 56 74 14 5,860 50 58 95 15 13,000 70 82 283 24 8,410 83 378 46725 18,300 188 381 1,360 27 3,790 440 881 375 35 5,940 230 814 516^(a)Values are from single oral cassette dosing at 1 mg/kg in 120-150 μlof PEG400 in triglyceride

TABLE 6 Optimum Mass Spectrometer Conditions and Key Fragmentation ofthe sEH Inhibitors Precursor Compound ions Predominant CXP ID m/z [M +H]⁺ product ion m/z DP (V) CE (V) (V) CUDA^(a) 341.2 216.2 76 25 10 2334.2 157.1 51 15 6 3 296.1 157.1 51 21 8 4 282.1 157.1 61 19 12 12290.1 183.1 56 19 14 13 304.4 122.1 56 29 6 14 318.1 183.1 51 21 14 15305.8 84.1 51 55 4 24 293.7 183.1 31 19 8 25 310.0 183.0 66 21 16 27402.1 183.1 77 19 12 35 378.1 183.1 66 23 12 APAU^(b) 320.2 143.1 71 1910 AUDA 393.2 135.1 81 37 10^(a)12-(3-cyclobexan-1-yl-ureido)-dodecanoic acid (CUDA) was used as aninternal standard to track instrument stability.^(b)1-(1-acetypiperidin-4-yl)-3-admantanylurea (APAU) was used as aninternal standard added after thawing samples but before extraction totrack the extraction efficiency of structurally similar target analytes.

TABLE 7 Cumulative Table of Inhibitor Structures, Results and PropertiesCompound IC₅₀ (nM) log P MP Synthetic ID Structure Human Murine (±0.5)(° C.) Method(s)  2

2.8 1.2 3.1 201-221 Ref. 12  3

3.9 0.9 2.3 164-172 B  4

12 3.5 1.8 177-179 B  5

3.2 0.4 3.5 131-132 A, B  6

2.7 7.4 1.8 155-158 B  7

130 49 1.3 169-171 B  8

3.0 4.2 2.4 213-215 A, B  9

3,800 12,000 <0 Oil B 10

1,700 5,100 1.6 178-183 A, B 11

40 8.7 1.8 173-175 A, B 12

43 55 1.8 180-182 A, B 13

8.3 1.3 2.3 164-165 A, B 14

2.8 3.3 2.8 173-174 A, B 15

87 8.7 1.0 164-165 A, B 16

3.5 0.4 2.8 153-154 A, B 17

61 100 1.1 195-197 C 18

>5,000 25,000 0.8 173-175 A, B 19

2,000 650 0.2 221-225 A, B 20

38 97 1.7 240-241 B 21

140 64 1.6 192-204 B 22

330 1,000 0.4 201-204 From 21 23

15 1,400 0.0 229-230 A, B 24

79 110 1.4 183-184 B 25

10 23 2.2 225-226 B 26

3.6 15 2.4 233-239 B 27

7.2 1.4 2.5 246-247 A, B 28

39 20 1.7 158-164 B 29

300 780 1.6 127-130 B 30

21 6.6 2.2 165-166 B 31

1100 2900 2.0 150-157 C 32

3.4 0.6 2.9 198-200 A, B 33

0.4 1.0 3.3 196-198 A, B 34

>50,000 38,000 1.3 170-174 B 35

4.1 2.3 3.0 182-184 B 36

0.7 6.5 2.4 224-228 B 37

17 8.8 2.4 153-154 B 38

0.4 0.7 3.5 160-164 A, B 39

17 28 3.8 226-229 A, B 40

3.7 2.8 2.5 195-196 B 47

0.7 1.3 2.4 210-212 D 48

0.6 0.7 2.9 208-209 D 49

3.1 5.0 2.6 183-187 D 50

1.5 18 3.8 178-183 D 51

0.5 1.2 2.4 111-122 From 50 52

0.4 0.4 2.7 195-196 D 53

0.4 0.4 3.1 150-154 See text 54

0.5 2.7 2.0 168-175 D 55

2.9 2.0 2.2 233-234 E 56

0.4 0.7 2.6 235-239 E 57

1.8 0.4 3.1 188-189 E 58

0.4 0.4 3.5 205-207 E 59

0.8 nd^(a) 4.3 102-107 E ^(a)nd = Not Determined

TABLE 8 Haloalkyl-Phenyl Ureas IC₅₀ IC₅₀ Compound Structure Name (FA¹)(tDPPO²) 36

1-(1-propionylpiperidin-4-yl)- 3-(4- (trifluoromethyl)phenyl)urea 9.389.6 60

1-(1-isobutyrylpiperidin-4-yl)- 3-(4- (trifluoromethyl)phenyl)urea 5.537.5 61

1-(1- (cyclopropanecarbonyl) piperidin- 4-yl)-3-(4-(trifluoromethyl)phenyl)urea 1.7 22.6 62

1-(4-(trifluoromethyl)phenyl)-3- (1-(3,3,3-trifluoropropionoyl)piperidin-4- yl)urea 1.5 9.1 63

1-(1-butyrylpiperidin-4-yl)-3- (4-trifluoromethyl)phenyl)urea 7.9 63.864

1-(1-(2- methylbutanoyl)piperidin-4-yl)- 3-(4-(trifluoromethyl)phenyl)urea 4 17.8 65

1-(1-4,4,4- trifluorobutanoyl)piperidin-4- yl)-3-(4-(trifluoromethyl)phenyl)urea 9.3 70.8 66

1-(1-methylsulfonyl)piperidin- 4-yl)-3-(4- (trifluoromethyl)phenyl)urea28.9 148.0 67

1-(1-(ethylsulfonyl)piperidin-4- yl)-3-(4- (trifluoromethyl)phenyl)urea11.0 93.9 68

1-(1-(propylsulfonyl)piperidin- 4-yl)-3-(4- (trifluoromethyl)phenyl)urea6.0 45.4 69

1-(1-(butylsulfonyl)piperidin-4- yl)-3-(4- (trifluoromethyl)phenyl)urea6.8 27.0 70

1-(1- (cyclopropylsulfonyl)piperidin- 4-yl)-3-(4-(trifluoromethyl)phenyl)urea 9.3 92.1 71

1-(1- (isopropylsulfonyl)piperidin-4- yl)-3-(4-(trifluoromethyl)phenyl)urea 10.1 37.5 72

1-(1-((2,2,2- trifluoroethyl)sulfonyl)piperidin- 4-yl)-3-(4-(trifluoromethyl)phenyl)urea 3.6 23.6 73

1-(1-((3,3,3- trifluoropropyl)sulfonyl) piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea 74

1-(1- (cyclopropylsulfonyl)piperidin- 4-yl)-3-(4-isopropylphenyl)urea14.3 157.4 ¹FA is fluoresence competitive assay. ²t-DPPO istrans-diphenylpropene oxide, used in a radiometric competitive assay.

TABLE 9 Pharmacokinetic Screening Results for Selected Haloalkyl-PhenylUreas Inhibitors Tmax (h) Cmax (uM) T½ (h) AUC (uM * h) A (61) 4 10.210.6 170 B (70) 4 10.9 11.9 167 C (69) 1 1.7 4.5 15.5 D (64) 4 4.0 2.53.9

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

What is claimed is:
 1. A compound selected from the group consisting of:1-(4-Methoxyphenyl)-3-(1-propionylpiperidin-4-yl)urea,1-(4-Phenoxyphenyl)-3-(1-propionylpiperidin-4-y)urea,1-(4-Fluorophenyl)-3-(1-propionylpiperidin-4-yl)urea,1-(4-Chlorophenyl)-3-(1-propionylpiperidin-4-yl)urea,1-(4-Iodophenyl)-3-(1-propionylpiperidin-4-yl)urea,1-(3,5-Dichlorophenyl)-3-(1-propionylpiperidin-4-yl)urea,1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(1-propionylpiperidin-4-y)urea,1-(4-Perfluoroisopropylphenyl)-3-(1-propionylpiperidin-4-y)urea,1-(1-(2-methylbutyryl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea,1-(1-(3,3,3-trifluoropropionyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea,1-(1-(4,4,4-trifluorobutyryl)piperidin-4-yl)-3-(4-(trifluorometityl)phenyl)urea,1-(1-Propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea,1-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-3-(4-(trifluoromeithoxy)phenyl)urea,1-(1-(Trifluoroacetyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea,1-(1-(propylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea,1-(1-(butylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea,1-(1-(cyclopropylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea,1-(1-(2,2,2-trifluoroethylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea,1-(1-(3,3,3-trifluoropropylsulfonyppiperidin-4-yl)-3-(4-(triflubromethyl)phenyl)urea,1-(1-(Methylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea,and 1-(1-(Ethylsulfonyppiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea, or salts andisomers thereof.
 2. A pharmaceutical composition comprising a compoundof claim 1 and a pharmaceutically acceptable excipient.
 3. A method forinhibiting a soluble epoxide hydrolase, the method comprising contactingthe soluble epoxide hydrolase with an amount of a compound of claim 1sufficient to inhibit the soluble epoxide hydrolase.
 4. A method formonitoring the activity of a soluble epoxide hydrolase, the methodcomprising contacting the soluble epoxide hydrolase with an amount of acompound of claim 1 sufficient to produce a detectable change in thefluorescence of the soluble epoxide hydrolase by interacting with one ormore tryptophan residues present in the catalytic site of said sEH. 5.The compound of claim 1, wherein the compound is selected from the groupconsisting of:1-(1-cyclopropanecarbonylpiperidin-4-yl)-3-(4-trifluoromethyl)phenyl)urea,1-(1-(2-methylbutyryl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea,1-(1-(3,3,3-trifluoropropionyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea,and1-(1-(4,4,4-trifluorobutyryl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.6. The compound of claim 1, wherein the compound is selected from thegroup consisting of:1-(1-Propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea,1-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea,and1-(1-(Trifluoroacetyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.7. The compound of claim 1, wherein the compound is1-(1-cyclopropanecarbonylpiperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.8. The compound of claim 1, wherein the compound is selected from thegroup consisting of:1-(1-(propylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea,1-(1-(butylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea,1-(1-(cyclopropylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea,1-(1-(2,2,2-trifluoroethylsulfonyl)piperidin-4-yl)-3-(4-(trifluromethyl)phenyl)urea,and1-(1-(3,3,3-trifluoropropylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea.9. The compound of claim 1, wherein the compound is selected from thegroup consisting of:1-(1-(Methylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea,and1-(1-(Ethylsulfonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.10. A method for inhibiting a soluble epoxide hydrolase, the methodcomprising contacting the soluble epoxide hydrolase with an amount of1-(1-cyclopropanecarbonylpiperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureasufficient to inhibit the soluble epoxide hydrolase.
 11. A method formonitoring the activity of a soluble epoxide hydrolase, the methodcomprising contacting the soluble epoxide hydrolase with an amount of1-(1-cyclopropanecarbonylpiperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)ureasufficient to produce a detectable change in the fluorescence of thesoluble epoxide hydrolase by interacting with one or more tryptophanresidues present in the catalytic site of said sEH.